N-azaspirocycloalkane substituted n-heteroaryl compounds and compositions for inhibiting the activity of shp2

ABSTRACT

The present invention relates to compounds of formula I:in which p, q, Y1, Y2, R1, R2a, R2b, R3a, R3b, R4a, R4b, R5a, R5b, R7 and R8 are defined in the Summary of the Invention; capable of inhibiting the activity of SHP2. The invention further provides a process for the preparation of compounds of the invention, pharmaceutical preparations comprising such compounds and methods of using such compounds and compositions in the management of diseases or disorders associated with the aberrant activity of SHP2.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. Continuation patent application of U.S.patent application Ser. No. 17/182,744, filed Feb. 23, 2021, which is acontinuation of U.S. patent application Ser. No. 16/418,978, filed May21, 2019 and issued on Apr. 6, 2021 as U.S. Pat. No. 10,968,235, whichis a divisional of U.S. patent application Ser. No. 15/899,821, filedFeb. 20, 2018 and issued on Jul. 2, 2019 as U.S. Pat. No. 10,336,774,which is a divisional application of U.S. patent application of Ser. No.15/110,511, filed Jul. 8, 2016 and issued on Sep. 18, 2018 as U.S. Pat.No. 10,077,276, which is a 371 U.S. national phase application ofinternational application number PCT/IB2015/050345 filed 16 Jan. 2015,which application claims the benefit of priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 61/928,754, filed 17Jan. 2014 and U.S. Provisional Patent Application No. 61/991,129, filed9 May, 2014. The disclosure of these applications are incorporatedherein by reference in their entirety and for all purposes.

SEQUENCE LISTING

The instant application contains a Sequence Listing, which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy is namedPAT056058-US-CNT03_SL.txt, created 22 Nov. 2021 (1385 bytes).

BACKGROUND Field of the Invention

The present invention relates to compounds capable of inhibiting theactivity of SHP2. The invention further provides a process for thepreparation of compounds of the invention, pharmaceutical preparationscomprising such compounds and methods of using such compounds andcompositions in the management of diseases or disorders associated withthe aberrant activity of SHP2.

Background of the Invention

The Src Homolgy-2 phosphatase (SHP2) is a non-receptor protein tyrosinephosphatase encoded by the PTPN11 gene that contributes to multiplecellular functions including proliferation, differentiation, cell cyclemaintenance and migration. SHP2 is involved in signaling through theRas-mitogen-activated protein kinase, the JAK-STAT or thephosphoinositol 3-kinase-AKT pathways.

SHP2 has two N-terminal Src homology 2 domains (N-SH2 and C-SH2), acatalytic domain (PTP), and a C-terminal tail. The two SH2 domainscontrol the subcellular localization and functional regulation of SHP2.The molecule exists in an inactive, self-inhibited conformationstabilized by a binding network involving residues from both the N-SH2and PTP domains. Stimulation by, for example, cytokines or growthfactors leads to exposure of the catalytic site resulting in enzymaticactivation of SHP2.

Mutations in the PTPN11 gene and subsequently in SHP2 have beenidentified in several human diseases, such as Noonan Syndrome, LeopardSyndrome, juvenile myelomonocytic leukemias, neuroblastoma, melanoma,acute myeloid leukemia and cancers of the breast, lung and colon. SHP2,therefore, represents a highly attractive target for the development ofnovel therapies for the treatment of various diseases. The compounds ofthe present invention fulfill the need of small molecules to thatinhibit the activity of SHP2.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides compounds of Formula I.

in which: p is selected from 0 and 1; q is selected from 0 and 1; Y₁ isselected from CH and N; Y₂ is selected from CR₆ and N; R₁ is —XR_(1a);wherein R_(1a) is selected from C₆₋₁₀aryl, C₃₋₈cycloalkyl,C₃₋₈cycloalkenyl and a 5-9 member heteroaryl group containing from 1 to4 heteroatoms or groups independently selected from N, C(O), O and S;wherein said aryl or heteroaryl of R_(1a) is substituted with 1 to 5 R₉groups independently selected from halo, amino, hydroxy, N₃, C₁₋₄alkyl,dimethyl-amino, hydroxy-substituted-C₁₋₄alkyl,halo-substituted-C₁₋₄alkyl, amino-substituted-C₁₋₄alkyl, —C(O)OR₁₀ and—NHC(O)R₁₀; and X is selected from a bond, S(O)_(m), O, C(O), COR₁₁,CR_(10a)R_(10b), NR₁₁; wherein m is selected from 0, 1 and 2; eachR_(10a) and R_(10b) is independently selected from halo and C₁₋₄alkyl;and R₁₁ is selected from hydrogen and C₁₋₄alkyl; R_(2a) and R_(2b) areindependently selected from hydrogen, C₁₋₄alkyl, C₁₋₄alkoxy, amino,hydroxy, C₃₋₈cycloalkyl and C₁₋₄alkyl-amino; R_(3a) and R_(3b) areindependently selected from halo, carbonyl, C₁₋₄alkyl, C₁₋₄alkoxy,amino, hydroxy, C₃₋₈cycloalkyl and C₁₋₄alkyl-amino; R_(4a) and R_(4b)are independently selected from hydrogen, halo, carbonyl, C₁₋₄alkyl,C₁₋₄alkoxy, amino, hydroxy, C₃₋₈cycloalkyl and C₁₋₄alkyl-amino; R_(5a)and R_(5b) are independently selected from hydrogen, carbonyl,C₁₋₄alkyl, C₁₋₄alkoxy, amino, hydroxy, C₃₋₈cycloalkyl andC₁₋₄alkyl-amino; wherein any two groups selected from R_(2a), R_(2b),R_(3a), R_(3b), R_(4a), R_(4b), R_(5a), R_(5b) and R₇ can form a 5 to 6member unsaturated or partially saturated ring; R₆ is selected fromhydrogen, halo, cyano, C₁₋₄alkyl, C₁₋₄alkoxy, amino-carbonyl,halo-substituted C₁₋₄alkyl, halo-substituted C₁₋₄alkoxy,hydroxy-substituted C₁₋₄alkyl, amino-substituted C₁₋₄alkyl,—S(O)₁₋₂R_(6a), —C(S)R_(6a), —C(O)NR_(6a)R_(6b), —C(NH)NR_(6a)R_(6b) and—NR_(6a)C(O)R_(6b); wherein R_(6a) and R_(6b) are independently selectedfrom hydrogen and C₁₋₄alkyl; R₇ and R₈ together with the carbon atom towhich they are both attached form a 3 to 7 member saturated or partiallyunsaturated ring that can optionally contain 1 to 3 heteroatoms orgroups independently selected from N, C(O), O and S(O)m; wherein m isselected from 0, 1 and 2; wherein said saturated ring formed by R₇ andR₈ can be unsubstituted or substituted with 1 to 3 groups independentlyselected from amino, hydroxy, methoxy, halo, methyl, methyl-amino andisobutyryloxy.

In a second aspect, the present invention provides a pharmaceuticalcomposition which contains a compound of Formula I or a N-oxidederivative, tautomer, individual isomers and mixture of isomers thereof;or a pharmaceutically acceptable salt thereof, in admixture with one ormore suitable excipients.

In a third aspect, the present invention provides a method of treating adisease in an animal in which modulation of SHP2 activity can prevent,inhibit or ameliorate the pathology and/or symptomology of the diseases,which method comprises administering to the animal a therapeuticallyeffective amount of a compound of Formula I or a N-oxide derivative,individual isomers and mixture of isomers thereof, or a pharmaceuticallyacceptable salt thereof.

In a fourth aspect, the present invention provides a method of treatinga disease in an animal in which modulation of SHP2 activity can prevent,inhibit or ameliorate the pathology and/or symptomology of the diseases,which method comprises administering to the animal a therapeuticallyeffective amount of a compound of Formula I or a N-oxide derivative,individual isomers and mixture of isomers thereof, or a pharmaceuticallyacceptable salt thereof, in simultaneous or sequential combination withan anti-cancer therapeutic.

In a fifth aspect, the present invention provides the use of a compoundof Formula I in the manufacture of a medicament for treating a diseasein an animal in which SHP2 activity contributes to the pathology and/orsymptomology of the disease.

In a sixth aspect, the present invention provides a process forpreparing compounds of Formula I and the N-oxide derivatives, prodrugderivatives, protected derivatives, individual isomers and mixture ofisomers thereof, and the pharmaceutically acceptable salts thereof.

Definitions

The general terms used hereinbefore and hereinafter preferably havewithin the context of this disclosure the following meanings, unlessotherwise indicated, where more general terms wherever used may,independently of each other, be replaced by more specific definitions orremain, thus defining more detailed embodiments of the invention:

“Alkyl” refers to a fully saturated branched or unbranched hydrocarbonmoiety having up to 20 carbon atoms. Unless otherwise provided, alkylrefers to hydrocarbon moieties having 1 to 7 carbon atoms (C₁₋₇alkyl),or 1 to 4 carbon atoms (C₁₋₄alkyl). Representative examples of alkylinclude, but are not limited to, methyl, ethyl, n-propyl, iso-propyl,n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl,neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl,2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl and the like. Asubstituted alkyl is an alkyl group containing one or more, such as one,two or three substituents selected from halogen, hydroxy or alkoxygroups. Halo-substituted-alkyl and halo-substituted-alkoxy, can beeither straight-chained or branched and includes, methoxy, ethoxy,difluoromethyl, trifluoromethyl, pentafluoroethyl, difluoromethoxy,trifluoromethoxy, and the like.

“Aryl” means a monocyclic or fused bicyclic aromatic ring assemblycontaining six to ten ring carbon atoms. For example, aryl may be phenylor naphthyl, preferably phenyl. “Arylene” means a divalent radicalderived from an aryl group.

“Heteroaryl” is as defined for aryl above where one or more of the ringmembers is a heteroatom. For example C₅₋₁₀heteroaryl is a minimum of 5members as indicated by the carbon atoms but that these carbon atoms canbe replaced by a heteroatom. Consequently, C₅₋₁₀heteroaryl includespyridyl, indolyl, indazolyl, quinoxalinyl, quinolinyl, benzofuranyl,benzopyranyl, benzothiopyranyl, benzo[1,3]dioxole, imidazolyl,benzo-imidazolyl, pyrimidinyl, furanyl, oxazolyl, isoxazolyl, triazolyl,tetrazolyl, pyrazolyl, thienyl, etc.

“Cycloalkyl” means a saturated or partially unsaturated, monocyclic,fused bicyclic or bridged polycyclic ring assembly containing the numberof ring atoms indicated. For example, C₃₋₁₀cycloalkyl includescyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, etc.

“Heterocycloalkyl” means cycloalkyl, as defined in this application,provided that one or more of the ring carbons indicated, are replaced bya moiety selected from —O—, —N═, —NR—, —C(O)—, —S—, —S(O)— or —S(O)₂—,wherein R is hydrogen, C₁₋₄alkyl or a nitrogen protecting group. Forexample, C₃₋₈heterocycloalkyl as used in this application to describecompounds of the invention includes morpholino, pyrrolidinyl,pyrrolidinyl-2-one, piperazinyl, piperidinyl, piperidinylone,1,4-dioxa-8-aza-spiro[4.5]dec-8-yl, thiomorpholino, sulfanomorpholino,sulfonomorpholino, etc.

“Halogen” (or halo) preferably represents chloro or fluoro, but may alsobe bromo or iodo.

“SHP2” means “Src Homolgy-2 phosphatase” and is also known as SH-PTP2,SH-PTP3, Syp, PTP1D, PTP2C, SAP-2 or PTPN11.

Cancers harboring “PTPN11 mutations” include but are not limited to:N58Y; D61Y, V; E69K; A72V, T, D; E76G, Q, K (ALL); G60A; D61Y; E69V;F71K; A72V; T73I; E76G, K; R289G; G503V (AML); G60R, D61Y, V, N; Y62D;E69K; A72T, V; T73I; E76K, V, G, A, Q; E139D; G503A, R; Q506P (JMML);G60V; D61V; E69K; F71L; A72V; E76A (MDS); Y63C (CMML); Y62C; E69K; T507K(neuroblastoma); V46L; N58S; E76V (Lung cancer); R138Q (melanoma); E76G(colon cancer).

Compounds of formula I may have different isomeric forms. For example,any asymmetric carbon atom may be present in the (R)-, (S)- or(R,S)-configuration, preferably in the (R)- or (S)-configuration.Substituents at a double bond or especially a ring may be present incis-(═Z—) or trans (=E-) form. The compounds may thus be present asmixtures of isomers or preferably as pure isomers, preferably as purediastereomers or pure enantiomers.

Where the plural form (e.g. compounds, salts) is used, this includes thesingular (e.g. a single compound, a single salt). “A compound” does notexclude that (e.g. in a pharmaceutical formulation) more than onecompound of the formula I (or a salt thereof) is present, the “a” merelyrepresenting the indefinite article. “A” can thus preferably be read as“one or more”, less preferably alternatively as “one”.

Wherever a compound or compounds of the formula I are mentioned, this isfurther also intended to include N-oxides of such compounds and/ortautomers thereof.

The term “and/or an N-oxide thereof, a tautomer thereof and/or a(preferably pharmaceutically acceptable) salt thereof” especially meansthat a compound of the formula I may be present as such or in mixturewith its N-oxide, as tautomer (e.g. due to keto-enol, lactam-lactim,amide-imidic acid or enamine-imine tautomerism) or in (e.g. equivalencyreaction caused) mixture with its tautomer, or as a salt of the compoundof the formula I and/or any of these forms or mixtures of two or more ofsuch forms.

For the following compounds, the NH2 group attached to the pyrazine ringis critical for potency. Analysis of the crystallographic structureshows the NH2 group in an intramolecular interaction with the backbonecarbonyl group of SHP2 residue E250:

Compound SHP2 IC50

70 nM

5.7 μM

The present invention also includes all suitable isotopic variations ofthe compounds of the invention, or pharmaceutically acceptable saltsthereof. An isotopic variation of a compound of the invention or apharmaceutically acceptable salt thereof is defined as one in which atleast one atom is replaced by an atom having the same atomic number butan atomic mass different from the atomic mass usually found in nature.Examples of isotopes that may be incorporated into the compounds of theinvention and pharmaceutically acceptable salts thereof include, but arenot limited to, isotopes of hydrogen, carbon, nitrogen and oxygen suchas as ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ¹⁵N, ¹⁷O, ¹⁸O, ³⁵S, ¹⁸F, ³⁶Cl and ¹²³I.Certain isotopic variations of the compounds of the invention andpharmaceutically acceptable salts thereof, for example, those in which aradioactive isotope such as ³H or ¹⁴C is incorporated, are useful indrug and/or substrate tissue distribution studies. In particularexamples, ³H and ¹⁴C isotopes may be used for their ease of preparationand detectability. In other examples, substitution with isotopes such as²H may afford certain therapeutic advantages resulting from greatermetabolic stability, such as increased in vivo half-life or reduceddosage requirements. Isotopic variations of the compounds of theinvention or pharmaceutically acceptable salts thereof can generally beprepared by conventional procedures using appropriate isotopicvariations of suitable reagents. For example, a compound of theinvention can exist in a deutorated form as shown below:

DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention relates to compounds capable of inhibiting theactivity of SHP2. In one aspect of the invention, with respect tocompounds of formula I, —XR_(1a) is —SR_(1a) and is selected from:

In another aspect of the invention, —XR_(1a) is —SR_(1a) and is selectedfrom:

In another aspect of the invention, with respect to compounds of formulaI:

is selected from:

In another aspect, with respect to compounds of Formula I, are compoundsof Formula Ia:

in which: n is selected from 1, 2, 3 and 4; p is selected from 0 and 1;q is selected from 0 and 1; Y₁ is selected from CH and N; Y₂ is selectedfrom CR₆ and N; Y₄ is independently selected from N, C(O) and CR₉;wherein only one Y₄ is C(O); R₆ is selected from hydrogen, halo, methyland amino-carbonyl; R₇ and R₈ together with the carbon atom to whichthey are both attached form a 3 to 7 member saturated or partiallyunsaturated ring that can optionally contain a heteroatom selected fromN, O and S(O)m; wherein m is selected from 0, 1 and 2; wherein saidsaturated ring formed by R₇ and R₈ can be unsubstituted or substitutedwith a group selected from amino, amino-methyl and methyl-amino; R₉ isselected from halo, amino, dimethyl-amino, hydroxy, N₃, C₁₋₄alkyl,halo-substituted-C₁₋₄alkyl, C₁₋₄alkoxy, —C(O)OR₁₀ and —NHC(O)R₁₀; R₁₀ isselected from hydrogen, phenyl and naphthyl; wherein said phenyl of R₁₀is unsubstituted or substituted with methoxy; or a pharmaceuticallyacceptable salt thereof.

In a further aspect of the invention, R₇ and R₈ together with the carbonatom to which they are both attached form a 5 member saturated orpartially unsaturated ring that can optionally contain 1 to 2heteroatoms or groups independently selected from N, O, C(O) and S(O)m;wherein m is selected from 0, 1 and 2; wherein said saturated ringformed by R₇ and R₈ is substituted with 1 to 3 groups independentlyselected from amino, hydroxy, methoxy, halo, methyl, methyl-amino andisobutyryloxy; or a pharmaceutically acceptable salt thereof.

In a further aspect of the invention are compounds, or thepharmaceutically acceptable salt thereof, selected from:

In another aspect of the invention are compounds in which R₇ and R₈together with the carbon atom to which they are both attached form a 6member saturated or partially unsaturated ring that can optionallycontain a heteroatom selected from N, O and S(O)m; wherein m is selectedfrom 0, 1 and 2; wherein said saturated ring formed by R₇ and R₈ issubstituted with a group selected from amino, amino-methyl andmethyl-amino; or a pharmaceutically acceptable salt thereof.

In a further aspect of the invention are compounds, or thepharmaceutically acceptable salt thereof, selected from:

In another aspect of the invention are compounds in which R₇ and R₈together with the carbon atom to which they are both attached form a 4member saturated or partially unsaturated ring that can optionallycontain a heteroatom selected from N, O and S(O)m; wherein m is selectedfrom 0, 1 and 2; wherein said saturated ring formed by R₇ and R₈ issubstituted with a group selected from amino, amino-methyl andmethyl-amino; or a pharmaceutically acceptable salt thereof.

In a further aspect of the invention are compounds, or thepharmaceutically acceptable salt thereof, selected from:

In another aspect of the invention are compounds in which p and q areboth 0; or the pharmaceutically acceptable salt thereof.

In a further aspect of the invention are compounds, or thepharmaceutically acceptable salt thereof, selected from:

In another aspect of the invention are compounds of formula II:

in which: p is selected from 0 and 1; q is selected from 0 and 1; Y₁ isselected from CH and N; Y₂ is selected from CR₆ and N; R₁ is selectedfrom C₆₋₁₀aryl, C₃₋₈cycloalkyl, C₃₋₈cycloalkenyl and a 5-9 memberheteroaryl group containing from 1 to 4 heteroatoms selected from N, Oand S; wherein said aryl or heteroaryl of R_(1a) is substituted with 1to 5 R₉ groups independently selected from halo, amino, hydroxy, N₃,C₁₋₄alkyl, hydroxy-substituted-C₁₋₄alkyl, halo-substituted-C₁₋₄alkyl,amino-substituted-C₁₋₄alkyl, —C(O)OR₁₀ and —NHC(O)R₁₀; wherein m isselected from 0, 1 and 2; each R_(10a) and R_(10b) is independentlyselected from halo and C₁₋₄alkyl; and R₁₁ is selected from hydrogen andC₁₋₄alkyl; R_(2a) and R_(2b) are independently selected from hydrogen,C₁₋₄alkyl, C₁₋₄alkoxy, amino, hydroxy, C₃₋₈cycloalkyl andC₁₋₄alkyl-amino; R_(3a) and R_(3b) are independently selected from halo,carbonyl, C₁₋₄alkyl, C₁₋₄alkoxy, amino, hydroxy, C₃₋₈cycloalkyl andC₁₋₄alkyl-amino; R_(4a) and R_(4b) are independently selected fromhydrogen, halo, carbonyl, C₁₋₄alkyl, C₁₋₄alkoxy, amino, hydroxy,C₃₋₈cycloalkyl and C₁₋₄alkyl-amino; R_(5a) and R_(5b) are independentlyselected from hydrogen, carbonyl, C₁₋₄alkyl, C₁₋₄alkoxy, amino, hydroxy,C₃₋₈cycloalkyl and C₁₋₄alkyl-amino; wherein any two groups selected fromR_(2a), R_(3a), R₄, R₅, R_(6a) and R_(7a) can form a 5 to 6 memberunsaturated or partially unsaturated ring; R₆ is selected from hydrogen,halo, cyano, C₁₋₄alkyl, C₁₋₄alkoxy, amino-carbonyl, halo-substitutedC₁₋₄alkyl, halo-substituted C₁₋₄alkoxy, hydroxy-substituted C₁₋₄alkyland amino-substituted C₁₋₄alkyl; R₇ and R₈ together with the carbon atomto which they are both attached form a 3 to 7 member saturated orpartially unsaturated ring that can optionally contain a heteroatomselected from N, O and S(O)m; wherein m is selected from 0, 1 and 2;wherein said saturated ring formed by R₇ and R₈ can be unsubstituted orsubstituted with a group selected from amino, amino-methyl andmethyl-amino; or a pharmaceutically acceptable salt thereof.

In a further aspect of the invention are compounds of formula IIa:

in which: n is selected from 1, 2, 3 and 4; p is selected from 0 and 1;q is selected from 0 and 1; Y₁ is selected from CH and N; Y₂ is selectedfrom CR₆ and N; Y₄ is selected from N and CR₉; R₆ is selected fromhydrogen, halo, methyl and amino-carbonyl; R₇ and R₈ together with thecarbon atom to which they are both attached form a 3 to 7 membersaturated or partially unsaturated ring that can optionally contain aheteroatom selected from N, O and S(O)m; wherein m is selected from 0, 1and 2; wherein said saturated ring formed by R₇ and R₈ can beunsubstituted or substituted with a group selected from amino,amino-methyl and methyl-amino; R₉ is selected from halo, amino, hydroxy,N₃, C₁₋₄alkyl, halo-substituted-C₁₋₄alkyl, C₁₋₄alkoxy, —C(O)OR₁₀ and—NHC(O)R₁₀; R₁₀ is selected from hydrogen, phenyl and naphthyl; whereinsaid phenyl of R₁₀ is unsubstituted or substituted with methoxy; or apharmaceutically acceptable salt thereof.

In a further aspect of the invention are compounds in which R₇ and R₈together with the carbon atom to which they are both attached form a 5member saturated ring that can optionally contain a heteroatom selectedfrom N, O and S(O)m; wherein m is selected from 0, 1 and 2; wherein saidsaturated ring formed by R₇ and R₈ is substituted with a group selectedfrom amino, amino-methyl and methyl-amino; or a pharmaceuticallyacceptable salt thereof.

In a further aspect of the invention are compounds, or thepharmaceutically acceptable salt thereof, selected from:

In another aspect of the invention are compounds in which R₇ and R₈together with the carbon atom to which they are both attached form a 6member saturated ring that can optionally contain a heteroatom selectedfrom N, O and S(O)m; wherein m is selected from 0, 1 and 2; wherein saidsaturated ring formed by R₇ and R₈ is substituted with a group selectedfrom amino, amino-methyl and methyl-amino; or a pharmaceuticallyacceptable salt thereof.

In a further aspect of the invention are compounds, or thepharmaceutically acceptable salt thereof, selected from:

In another aspect of the invention are compounds in which R₇ and R₈together with the carbon atom to which they are both attached form a 4member saturated ring that can optionally contain a heteroatom selectedfrom N, O and S(O)m; wherein m is selected from 0, 1 and 2; wherein saidsaturated ring formed by R₇ and R₈ is substituted with amino; or apharmaceutically acceptable salt thereof.

In a further aspect of the invention are compounds, or thepharmaceutically acceptable salt thereof, selected from:

In another aspect of the invention are compounds in which p and q areboth 0; or the pharmaceutically acceptable salt thereof.

In a further aspect of the invention are compounds, or thepharmaceutically acceptable salt thereof, selected from:

Pharmacology and Utility

The Src Homolgy-2 phosphatase (SHP2) is a protein tyrosine phosphataseencoded by the PTPN11 gene that contributes to multiple cellularfunctions including proliferation, differentiation, cell cyclemaintenance and migration. SHP2 is involved in signaling through theRas-mitogen-activated protein kinase, the JAK-STAT or thephosphoinositol 3-kinase-AKT pathways. SHP2 mediates activation of Erk1and Erk2 (Erk1/2, Erk) MAP kinases by receptor tyrosine kinases such asErbB1, ErbB2 and c-Met.

SHP2 has two N-terminal Src homology 2 domains (N-SH2 and C-SH2), acatalytic domain (PTP), and a C-terminal tail. The two SH2 domainscontrol the subcellular localization and functional regulation of SHP2.The molecule exists in an inactive conformation, inhibiting its ownactivity via a binding network involving residues from both the N-SH2and PTP domains. In response to growth factor stimulation, SHP2 binds tospecific tyrosine-phosphorylated sites on docking proteins such as Gab1and Gab2 via its SH2 domains. This induces a conformational change thatresults in SHP2 activation.

Mutations in PTPN11 have been identified in several human diseases, suchas Noonan Syndrome, Leopard Syndrome, juvenile myelomonocytic leukemias,neuroblastoma, melanoma, acute myeloid leukemia and cancers of thebreast, lung and colon. SHP2 is an important downstream signalingmolecule for a variety of receptor tyrosine kinases, including thereceptors of platelet-derived growth factor (PDGF-R), fibroblast growthfactor (FGF-R) and epidermal growth factor (EGF-R). SHP2 is also animportant downstream signaling molecule for the activation of themitogen activated protein (MAP) kinase pathway which can lead to celltransformation, a prerequisite for the development of cancer. Knock-downof SHP2 significantly inhibited cell growth of lung cancer cell lineswith SHP2 mutation or EML4/ALK translocations as well as EGFR amplifiedbreast cancers and esophageal cancers. SHP2 is also activated downstreamof oncogenes in gastric carcinoma, anaplastic large-cell lymphoma andglioblastoma.

Noonan Syndrome (NS) and Leopard Syndrome (LS)—PTPN11 mutations cause LS(multiple lentigenes, electrocardiographic conduction abnormalities,ocular hypertelorism, pulmonic stenosis, abnormal genitalia, retardationof growth, sensorineural deafness) and NS (congenital anomaliesincluding cardiac defects, craniofacial abnormalities and shortstature). Both disorders are part of a family of autosomal dominantsyndromes caused by germline mutations in components of theRAS/RAF/MEK/ERK mitogen activating protein kinase pathway, required fornormal cell growth and differentiation. Aberrant regulation of thispathway has profound effects, particularly on cardiac development,resulting in various abnormalities, including valvuloseptal defectsand/or hypertrophic cardiomyopathy (HCM). Perturbations of the MAPKsignaling pathway have been established as central to these disordersand several candidate genes along this pathway have been identified inhumans, including mutations in KRAS, NRAS, SOS1, RAF1, BRAF, MEK1, MEK2,SHOC2, and CBL. The gene most commonly mutated in NS and LS is PTPN11.Germline mutations in PTPN11 (SHP2) are found in ˜50% of the cases withNS and nearly all patients with LS that shares certain features with NS.For NS, Y62D and Y63C substitutions in the protein are largely invariantand are among the most common mutations. Both these mutations affect thecatalytically inactive conformation of SHP2 without perturbing thebinding of the phosphatase to its phosphorylated signaling partners.

Juvenile Myelomonocytic Leukemias (JMML)—Somatic mutations in PTPN11(SHP2) occur in about 35% of the patients with JMML, a childhoodmyeloproliferative disorder (MPD). These gain-of-function mutations aretypically point mutations in the N-SH2 domain or in the phosphatasedomain, which prevent self-inhibition between the catalytic domain andthe N-SH2 domain, resulting in SHP2 activity.

Acute Myeloid Leukemia—PTPN11 mutations have been identified in: ˜10% ofpediatric acute leukemias, such as myelodysplastic syndrome (MDS); ˜7%of B cell acute lymphoblastic leukemia (B-ALL); and ˜4% of acute myeloidleukemia (AML).

NS and leukemia mutations cause changes in amino acids located at theinterface formed by the N-SH2 and PTP domains in the self-inhibited SHP2conformation, disrupting the inhibitory intramolecular interaction,leading to hyperactivity of the catalytic domain.

SHP2 acts as a positive regulator in receptor tyrosine kinase (RTK)signaling. Cancers containing RTK alterations (EGFR^(amp), Her2^(amp),FGFR^(amp), Met^(amp), translocated/activated RTK, i.e. ALK, BCR/ABL)include Esophageal, Breast, Lung, Colon, Gastric, Glioma, Head and Neckcancers.

Esophageal cancer (or oesophageal cancer) is a malignancy of theesophagus. There are various subtypes, primarily squamous cell cancer(<50%) and adenocarcinoma. There is a high rate of RTK expression inesophageal adenocarcinoma and squamous cell cancer. A SHP2 inhibitor ofthe invention can, therefore, be employed for innovative treatmentstrategies.

Breast cancer is a major type of cancer and a leading cause of death inwomen, where patients develop resistance to current drugs. There arefour major subtypes of breast cancers including luminal A, luminal B,Her2 like, and triple negative/Basal-like. Triple negative breast cancer(TNBC) is an aggressive breast cancer lacking specific targeted therapy.Epidermal growth factor receptor I (EGFR) has emerged as a promisingtarget in TNBC. Inhibition of Her2 as well as EGFR via SHP2 may be apromising therapy in breast cancer.

Lung Cancer—NSCLC is currently a major cause of cancer-relatedmortality. accounting for about 85% of lung cancers (predominantlyadenocarcinomas and squamous cell carcinomas). Although cytotoxicchemotherapy remains an important part of treatment, targeted therapiesbased on genetic alterations such as EGFR and ALK in the tumor are morelikely to benefit from a targeted therapy.

Colon Cancer—Approximately 30% to 50% of colorectal tumors are known tohave a mutated (abnormal) KRAS, and BRAF mutations occur in 10 to 15% ofcolorectal cancers. For a subset of patients whose colorectal tumorshave been demonstrated to over express EGFR, these patients exhibit afavorable clinical response to anti-EGFR therapy.

Gastic Cancer is one of the most prevalent cancer types. Aberrantexpression of tyrosine kinases, as reflected by the aberrant tyrosinephosphorylation in gastric cancer cells, is known in the art. Threereceptor-tyrosine kinases, c-met (HGF receptor), FGF receptor 2, anderbB2 neu are frequently amplified in gastric carcinomas. Thus,subversion of different signal pathways may contribute to theprogression of different types of gastric cancers.

Neuroblastoma is a pediatric tumor of the developing sympathetic nervoussystem, accounting for about 8% of childhood cancers. Genomicalterations of the anaplastic lymphoma kinase (ALK) gene have beenpostulated to contribute to neuroblastoma pathogenesis.

Squamous-cell carcinoma of the head and neck (SCCHN). High levels ofEGFR expression are correlated with poor prognosis and resistance toradiation therapy in a variety of cancers, mostly in squamous-cellcarcinoma of the head and neck (SCCHN). Blocking of the EGFR signalingresults in inhibition of the stimulation of the receptor, cellproliferation, and reduced invasiveness and metastases. The EGFR is,therefore, a prime target for new anticancer therapy in SCCHN.

The present invention relates to compounds capable of inhibiting theactivity of SHP2. The invention further provides a process for thepreparation of compounds of the invention and pharmaceuticalpreparations comprising such compounds. Another aspect of the presentinvention relates to a method of treating SHP2-mediated disorderscomprising the step of administering to a patient in need thereof atherapeutically effective amount of a compound of formula I as definedin the Summary of the Invention.

In certain embodiments, the present invention relates to theaforementioned method, wherein said SHP2-mediated disorders are cancersselected from, but not limited to: JMML; AML; MDS; B-ALL; neuroblastoma;esophageal; breast cancer; lung cancer; colon cancer; Gastric cancer,Head and Neck cancer.

The compounds of the present invention may also be useful in thetreatment of other diseases or conditions related to the aberrantactivity of SHP2. Thus, as a further aspect, the invention relates to amethod of treatment of a disorder selected from: NS; LS; JMML; AML; MDS;B-ALL; neuroblastoma; esophageal; breast cancer; lung cancer; coloncancer; gastric cancer; head and neck cancer.

A SHP2 inhibitor of the present invention may be usefully combined withanother pharmacologically active compound, or with two or more otherpharmacologically active compounds, particularly in the treatment ofcancer. For example, a compound of the formula (I), or apharmaceutically acceptable salt thereof, as defined above, may beadministered simultaneously, sequentially or separately in combinationwith one or more agents selected from chemotherapy agents, for example,mitotic inhibitors such as a taxane, a vinca alkaloid, paclitaxel,docetaxel, vincristine, vinblastine, vinorelbine or vinflunine, andother anticancer agents, e.g. cisplatin, 5-fluorouracil or5-fluoro-2-4(1H,3H)-pyrimidinedione (5FU), flutamide or gemcitabine.

Such combinations may offer significant advantages, includingsynergistic activity, in therapy.

In certain embodiments, the present invention relates to theaforementioned method, wherein said compound is administeredparenterally.

In certain embodiments, the present invention relates to theaforementioned method, wherein said compound is administeredintramuscularly, intravenously, subcutaneously, orally, pulmonary,intrathecally, topically or intranasally.

In certain embodiments, the present invention relates to theaforementioned method, wherein said compound is administeredsystemically.

In certain embodiments, the present invention relates to theaforementioned method, wherein said patient is a mammal.

In certain embodiments, the present invention relates to theaforementioned method, wherein said patient is a primate.

In certain embodiments, the present invention relates to theaforementioned method, wherein said patient is a human.

In another aspect, the present invention relates to a method of treatingan SHP2-mediated disorder, comprising the step of: administering to apatient in need thereof a therapeutically effective amount of achemothereutic agent in combination with a therapeutically effectiveamount of a compound of formula I as defined in the Summary of theInvention.

Pharmaceutical Compositions

In another aspect, the present invention provides pharmaceuticallyacceptable compositions which comprise a therapeutically-effectiveamount of one or more of the compounds described above, formulatedtogether with one or more pharmaceutically acceptable carriers(additives) and/or diluents. As described in detail below, thepharmaceutical compositions of the present invention may be speciallyformulated for administration in solid or liquid form, including thoseadapted for the following: (1) oral administration, for example,drenches (aqueous or non-aqueous solutions or suspensions), tablets,e.g., those targeted for buccal, sublingual, and systemic absorption,boluses, powders, granules, pastes for application to the tongue; (2)parenteral administration, for example, by subcutaneous, intramuscular,intravenous or epidural injection as, for example, a sterile solution orsuspension, or sustained-release formulation; (3) topical application,for example, as a cream, ointment, or a controlled-release patch orspray applied to the skin; (4) intravaginally or intrarectally, forexample, as a pessary, cream or foam; (5) sublingually; (6) ocularly;(7) transdermally; (8) nasally; (9) pulmonary; or (10) intrathecally.

The phrase “therapeutically-effective amount” as used herein means thatamount of a compound, material, or composition comprising a compound ofthe present invention which is effective for producing some desiredtherapeutic effect in at least a sub-population of cells in an animal ata reasonable benefit/risk ratio applicable to any medical treatment.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically-acceptable carrier” as used herein means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, manufacturing aid (e.g.,lubricant, talc magnesium, calcium or zinc stearate, or steric acid), orsolvent encapsulating material, involved in carrying or transporting thesubject compound from one organ, or portion of the body, to anotherorgan, or portion of the body. Each carrier must be “acceptable” in thesense of being compatible with the other ingredients of the formulationand not injurious to the patient. Some examples of materials which canserve as pharmaceutically-acceptable carriers include: (1) sugars, suchas lactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) pH buffered solutions; (21)polyesters, polycarbonates and/or polyanhydrides; and (22) othernon-toxic compatible substances employed in pharmaceutical formulations.

As set out above, certain embodiments of the present compounds maycontain a basic functional group, such as amino or alkylamino, and are,thus, capable of forming pharmaceutically-acceptable salts withpharmaceutically-acceptable acids. The term “pharmaceutically-acceptablesalts” in this respect, refers to the relatively non-toxic, inorganicand organic acid addition salts of compounds of the present invention.These salts can be prepared in situ in the administration vehicle or thedosage form manufacturing process, or by separately reacting a purifiedcompound of the invention in its free base form with a suitable organicor inorganic acid, and isolating the salt thus formed during subsequentpurification. Representative salts include the hydrobromide,hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate,valerate, oleate, palmitate, stearate, laurate, benzoate, lactate,phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate,napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonatesalts and the like. (See, for example, Berge et al. (1977)“Pharmaceutical Salts”, J. Pharm. Sci. 66:1-19).

The pharmaceutically acceptable salts of the subject compounds includethe conventional nontoxic salts or quaternary ammonium salts of thecompounds, e.g., from non-toxic organic or inorganic acids. For example,such conventional nontoxic salts include those derived from inorganicacids such as hydrochloride, hydrobromic, sulfuric, sulfamic,phosphoric, nitric, and the like; and the salts prepared from organicacids such as acetic, propionic, succinic, glycolic, stearic, lactic,malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic,phenylacetic, glutamic, benzoic, salicyclic, sulfanilic,2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, isothionic, and the like.

In other cases, the compounds of the present invention may contain oneor more acidic functional groups and, thus, are capable of formingpharmaceutically-acceptable salts with pharmaceutically-acceptablebases. The term “pharmaceutically-acceptable salts” in these instancesrefers to the relatively non-toxic, inorganic and organic base additionsalts of compounds of the present invention. These salts can likewise beprepared in situ in the administration vehicle or the dosage formmanufacturing process, or by separately reacting the purified compoundin its free acid form with a suitable base, such as the hydroxide,carbonate or bicarbonate of a pharmaceutically-acceptable metal cation,with ammonia, or with a pharmaceutically-acceptable organic primary,secondary or tertiary amine. Representative alkali or alkaline earthsalts include the lithium, sodium, potassium, calcium, magnesium, andaluminum salts and the like. Representative organic amines useful forthe formation of base addition salts include ethylamine, diethylamine,ethylenediamine, ethanolamine, diethanolamine, piperazine and the like.(See, for example, Berge et al., supra)

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically-acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Formulations of the present invention include those suitable for oral,nasal, topical (including buccal and sublingual), rectal, vaginal and/orparenteral administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any methods wellknown in the art of pharmacy. The amount of active ingredient which canbe combined with a carrier material to produce a single dosage form willvary depending upon the host being treated, the particular mode ofadministration. The amount of active ingredient which can be combinedwith a carrier material to produce a single dosage form will generallybe that amount of the compound which produces a therapeutic effect.Generally, out of one hundred percent, this amount will range from about0.1 percent to about ninety-nine percent of active ingredient,preferably from about 5 percent to about 70 percent, most preferablyfrom about 10 percent to about 30 percent.

In certain embodiments, a formulation of the present invention comprisesan excipient selected from the group consisting of cyclodextrins,celluloses, liposomes, micelle forming agents, e.g., bile acids, andpolymeric carriers, e.g., polyesters and polyanhydrides; and a compoundof the present invention. In certain embodiments, an aforementionedformulation renders orally bioavailable a compound of the presentinvention.

Methods of preparing these formulations or compositions include the stepof bringing into association a compound of the present invention withthe carrier and, optionally, one or more accessory ingredients. Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association a compound of the present invention withliquid carriers, or finely divided solid carriers, or both, and then, ifnecessary, shaping the product.

Formulations of the invention suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, lozenges (using aflavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) and/or as mouth washes and thelike, each containing a predetermined amount of a compound of thepresent invention as an active ingredient. A compound of the presentinvention may also be administered as a bolus, electuary or paste.

In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules, trouches and thelike), the active ingredient is mixed with one or morepharmaceutically-acceptable carriers, such as sodium citrate ordicalcium phosphate, and/or any of the following: (1) fillers orextenders, such as starches, lactose, sucrose, glucose, mannitol, and/orsilicic acid; (2) binders, such as, for example, carboxymethylcellulose,alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3)humectants, such as glycerol; (4) disintegrating agents, such asagar-agar, calcium carbonate, potato or tapioca starch, alginic acid,certain silicates, and sodium carbonate; (5) solution retarding agents,such as paraffin; (6) absorption accelerators, such as quaternaryammonium compounds and surfactants, such as poloxamer and sodium laurylsulfate; (7) wetting agents, such as, for example, cetyl alcohol,glycerol monostearate, and non-ionic surfactants; (8) absorbents, suchas kaolin and bentonite clay; (9) lubricants, such as talc, calciumstearate, magnesium stearate, solid polyethylene glycols, sodium laurylsulfate, zinc stearate, sodium stearate, stearic acid, and mixturesthereof; (10) coloring agents; and (11) controlled release agents suchas crospovidone or ethyl cellulose. In the case of capsules, tablets andpills, the pharmaceutical compositions may also comprise bufferingagents. Solid compositions of a similar type may also be employed asfillers in soft and hard-shelled gelatin capsules using such excipientsas lactose or milk sugars, as well as high molecular weight polyethyleneglycols and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present invention, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be formulated for rapid release,e.g., freeze-dried. They may be sterilized by, for example, filtrationthrough a bacteria-retaining filter, or by incorporating sterilizingagents in the form of sterile solid compositions which can be dissolvedin sterile water, or some other sterile injectable medium immediatelybefore use. These compositions may also optionally contain opacifyingagents and may be of a composition that they release the activeingredient(s) only, or preferentially, in a certain portion of thegastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions which can be used include polymeric substancesand waxes. The active ingredient can also be in micro-encapsulated form,if appropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration of the compounds of theinvention include pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. In addition to the activeingredient, the liquid dosage forms may contain inert diluents commonlyused in the art, such as, for example, water or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor and sesame oils),glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acidesters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

Formulations of the pharmaceutical compositions of the invention forrectal or vaginal administration may be presented as a suppository,which may be prepared by mixing one or more compounds of the inventionwith one or more suitable nonirritating excipients or carrierscomprising, for example, cocoa butter, polyethylene glycol, asuppository wax or a salicylate, and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the active compound.

Formulations of the present invention which are suitable for vaginaladministration also include pessaries, tampons, creams, gels, pastes,foams or spray formulations containing such carriers as are known in theart to be appropriate.

Dosage forms for the topical or transdermal administration of a compoundof this invention include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches and inhalants. The active compound maybe mixed under sterile conditions with a pharmaceutically-acceptablecarrier, and with any preservatives, buffers, or propellants which maybe required.

The ointments, pastes, creams and gels may contain, in addition to anactive compound of this invention, excipients, such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a compound of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of a compound of the present invention to the body. Such dosageforms can be made by dissolving or dispersing the compound in the propermedium. Absorption enhancers can also be used to increase the flux ofthe compound across the skin. The rate of such flux can be controlled byeither providing a rate controlling membrane or dispersing the compoundin a polymer matrix or gel.

Ophthalmic formulations, eye ointments, powders, solutions and the like,are also contemplated as being within the scope of this invention.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise one or more compounds of the invention incombination with one or more pharmaceutically-acceptable sterileisotonic aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containsugars, alcohols, antioxidants, buffers, bacteriostats, solutes whichrender the formulation isotonic with the blood of the intended recipientor suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms upon the subject compounds may be ensuredby the inclusion of various antibacterial and antifungal agents, forexample, paraben, chlorobutanol, phenol sorbic acid, and the like. Itmay also be desirable to include isotonic agents, such as sugars, sodiumchloride, and the like into the compositions. In addition, prolongedabsorption of the injectable pharmaceutical form may be brought about bythe inclusion of agents which delay absorption such as aluminummonostearate and gelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally-administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsule matrices ofthe subject compounds in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissue.

When the compounds of the present invention are administered aspharmaceuticals, to humans and animals, they can be given per se or as apharmaceutical composition containing, for example, 0.1 to 99% (morepreferably, 10 to 30%) of active ingredient in combination with apharmaceutically acceptable carrier.

The preparations of the present invention may be given orally,parenterally, topically, or rectally. They are of course given in formssuitable for each administration route. For example, they areadministered in tablets or capsule form, by injection, inhalation, eyelotion, ointment, suppository, etc. administration by injection,infusion or inhalation; topical by lotion or ointment; and rectal bysuppositories. Oral administrations are preferred.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular,subarachnoid, intraspinal and intrasternal injection and infusion.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a compound, drug or other materialother than directly into the central nervous system, such that it entersthe patient's system and, thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration.

These compounds may be administered to humans and other animals fortherapy by any suitable route of administration, including orally,nasally, as by, for example, a spray, rectally, intravaginally,parenterally, intracisternally and topically, as by powders, ointmentsor drops, including buccally and sublingually.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically-acceptable dosage forms by conventional methodsknown to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient which is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion ormetabolism of the particular compound being employed, the rate andextent of absorption, the duration of the treatment, other drugs,compounds and/or materials used in combination with the particularcompound employed, the age, sex, weight, condition, general health andprior medical history of the patient being treated, and like factorswell known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the compounds of the invention employed in thepharmaceutical composition at levels lower than that required in orderto achieve the desired therapeutic effect and gradually increase thedosage until the desired effect is achieved.

In general, a suitable daily dose of a compound of the invention will bethat amount of the compound which is the lowest dose effective toproduce a therapeutic effect. Such an effective dose will generallydepend upon the factors described above. Generally, oral, intravenous,intracerebroventricular and subcutaneous doses of the compounds of thisinvention for a patient, when used for the indicated analgesic effects,will range from about 0.0001 to about 100 mg per kilogram of body weightper day.

If desired, the effective daily dose of the active compound may beadministered as two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms. Preferred dosing is one administrationper day.

While it is possible for a compound of the present invention to beadministered alone, it is preferable to administer the compound as apharmaceutical formulation (composition).

The compounds according to the invention may be formulated foradministration in any convenient way for use in human or veterinarymedicine, by analogy with other pharmaceuticals.

In another aspect, the present invention provides pharmaceuticallyacceptable compositions which comprise a therapeutically-effectiveamount of one or more of the subject compounds, as described above,formulated together with one or more pharmaceutically acceptablecarriers (additives) and/or diluents. As described in detail below, thepharmaceutical compositions of the present invention may be speciallyformulated for administration in solid or liquid form, including thoseadapted for the following: (1) oral administration, for example,drenches (aqueous or non-aqueous solutions or suspensions), tablets,boluses, powders, granules, pastes for application to the tongue; (2)parenteral administration, for example, by subcutaneous, intramuscularor intravenous injection as, for example, a sterile solution orsuspension; (3) topical application, for example, as a cream, ointmentor spray applied to the skin, lungs, or mucous membranes; or (4)intravaginally or intrarectally, for example, as a pessary, cream orfoam; (5) sublingually or buccally; (6) ocularly; (7) transdermally; or(8) nasally.

The term “treatment” is intended to encompass also prophylaxis, therapyand cure.

The patient receiving this treatment is any animal in need, includingprimates, in particular humans, and other mammals such as equines,cattle, swine and sheep; and poultry and pets in general.

The compound of the invention can be administered as such or inadmixtures with pharmaceutically acceptable carriers and can also beadministered in conjunction with antimicrobial agents such aspenicillins, cephalosporins, aminoglycosides and glycopeptides.Conjunctive therapy, thus includes sequential, simultaneous and separateadministration of the active compound in a way that the therapeuticaleffects of the first administered one is not entirely disappeared whenthe subsequent is administered.

Microemulsification technology can improve bioavailability of somelipophilic (water insoluble) pharmaceutical agents. Examples includeTrimetrine (Dordunoo, S. K., et al., Drug Development and IndustrialPharmacy, 17(12), 1685-1713, 1991 and REV 5901 (Sheen, P. C., et al., JPharm Sci 80(7), 712-714, 1991). Among other things, microemulsificationprovides enhanced bioavailability by preferentially directing absorptionto the lymphatic system instead of the circulatory system, which therebybypasses the liver, and prevents destruction of the compounds in thehepatobiliary circulation.

While all suitable amphiphilic carriers are contemplated, the presentlypreferred carriers are generally those that haveGenerally-Recognized-as-Safe (GRAS) status, and that can both solubilizethe compound of the present invention and microemulsify it at a laterstage when the solution comes into a contact with a complex water phase(such as one found in human gastrointestinal tract). Usually,amphiphilic ingredients that satisfy these requirements have HLB(hydrophilic to lipophilic balance) values of 2-20, and their structurescontain straight chain aliphatic radicals in the range of C-6 to C-20.Examples are polyethylene-glycolized fatty glycerides and polyethyleneglycols.

Commercially available amphiphilic carriers are particularlycontemplated, including Gelucire-series, Labrafil, Labrasol, orLauroglycol (all manufactured and distributed by Gattefosse Corporation,Saint Priest, France), PEG-mono-oleate, PEG-di-oleate, PEG-mono-laurateand di-laurate, Lecithin, Polysorbate 80, etc (produced and distributedby a number of companies in USA and worldwide).

Hydrophilic polymers suitable for use in the present invention are thosewhich are readily water-soluble, can be covalently attached to avesicle-forming lipid, and which are tolerated in vivo without toxiceffects (i.e., are biocompatible). Suitable polymers includepolyethylene glycol (PEG), polylactic (also termed polylactide),polyglycolic acid (also termed polyglycolide), a polylactic-polyglycolicacid copolymer, and polyvinyl alcohol. Preferred polymers are thosehaving a molecular weight of from about 100 or 120 daltons up to about5,000 or 10,000 daltons, and more preferably from about 300 daltons toabout 5,000 daltons. In a particularly preferred embodiment, the polymeris polyethyleneglycol having a molecular weight of from about 100 toabout 5,000 daltons, and more preferably having a molecular weight offrom about 300 to about 5,000 daltons. In a particularly preferredembodiment, the polymer is polyethyleneglycol of 750 daltons (PEG(750)).Polymers may also be defined by the number of monomers therein; apreferred embodiment of the present invention utilizes polymers of atleast about three monomers, such PEG polymers consisting of threemonomers (approximately 150 daltons).

Other hydrophilic polymers which may be suitable for use in the presentinvention include polyvinylpyrrolidone, polymethoxazoline,polyethyloxazoline, polyhydroxypropyl methacrylamide,polymethacrylamide, polydimethylacrylamide, and derivatized cellulosessuch as hydroxymethylcellulose or hydroxyethylcellulose.

In certain embodiments, a formulation of the present invention comprisesa biocompatible polymer selected from the group consisting ofpolyamides, polycarbonates, polyalkylenes, polymers of acrylic andmethacrylic esters, polyvinyl polymers, polyglycolides, polysiloxanes,polyurethanes and co-polymers thereof, celluloses, polypropylene,polyethylenes, polystyrene, polymers of lactic acid and glycolic acid,polyanhydrides, poly(ortho)esters, poly(butic acid), poly(valeric acid),poly(lactide-co-caprolactone), polysaccharides, proteins, polyhyaluronicacids, polycyanoacrylates, and blends, mixtures, or copolymers thereof.

Cyclodextrins are cyclic oligosaccharides, consisting of 6, 7 or 8glucose units, designated by the Greek letter alpha, beta or gamma,respectively. Cyclodextrins with fewer than six glucose units are notknown to exist. The glucose units are linked by alpha-1,4-glucosidicbonds. As a consequence of the chair conformation of the sugar units,all secondary hydroxyl groups (at C-2, C-3) are located on one side ofthe ring, while all the primary hydroxyl groups at C-6 are situated onthe other side. As a result, the external faces are hydrophilic, makingthe cyclodextrins water-soluble. In contrast, the cavities of thecyclodextrins are hydrophobic, since they are lined by the hydrogen ofatoms C-3 and C-5, and by ether-like oxygens. These matrices allowcomplexation with a variety of relatively hydrophobic compounds,including, for instance, steroid compounds such as 17.beta.-estradiol(see, e.g., van Uden et al. Plant Cell Tiss. Org. Cult. 38:1-3-113(1994)). The complexation takes place by Van der Waals interactions andby hydrogen bond formation. For a general review of the chemistry ofcyclodextrins, see, Wenz, Agnew. Chem. Int. Ed. Engl., 33:803-822(1994).

The physico-chemical properties of the cyclodextrin derivatives dependstrongly on the kind and the degree of substitution. For example, theirsolubility in water ranges from insoluble (e.g.,triacetyl-beta-cyclodextrin) to 147% soluble (w/v)(G-2-beta-cyclodextrin). In addition, they are soluble in many organicsolvents. The properties of the cyclodextrins enable the control oversolubility of various formulation components by increasing or decreasingtheir solubility.

Numerous cyclodextrins and methods for their preparation have beendescribed. For example, Parmeter (I), et al. (U.S. Pat. No. 3,453,259)and Gramera, et al. (U.S. Pat. No. 3,459,731) described electroneutralcyclodextrins. Other derivatives include cyclodextrins with cationicproperties [Parmeter (II), U.S. Pat. No. 3,453,257], insolublecrosslinked cyclodextrins (Solms, U.S. Pat. No. 3,420,788), andcyclodextrins with anionic properties [Parmeter (III), U.S. Pat. No.3,426,011]. Among the cyclodextrin derivatives with anionic properties,carboxylic acids, phosphorous acids, phosphinous acids, phosphonicacids, phosphoric acids, thiophosphonic acids, thiosulphinic acids, andsulfonic acids have been appended to the parent cyclodextrin [see,Parmeter (III), supra]. Furthermore, sulfoalkyl ether cyclodextrinderivatives have been described by Stella, et al. (U.S. Pat. No.5,134,127).

Liposomes consist of at least one lipid bilayer membrane enclosing anaqueous internal compartment. Liposomes may be characterized by membranetype and by size. Small unilamellar vesicles (SUVs) have a singlemembrane and typically range between 0.02 and 0.05 m in diameter; largeunilamellar vesicles (LUVS) are typically larger than 0.05 mOligolamellar large vesicles and multilamellar vesicles have multiple,usually concentric, membrane layers and are typically larger than 0.1μm. Liposomes with several nonconcentric membranes, i.e., severalsmaller vesicles contained within a larger vesicle, are termedmultivesicular vesicles.

One aspect of the present invention relates to formulations comprisingliposomes containing a compound of the present invention, where theliposome membrane is formulated to provide a liposome with increasedcarrying capacity. Alternatively or in addition, the compound of thepresent invention may be contained within, or adsorbed onto, theliposome bilayer of the liposome. The compound of the present inventionmay be aggregated with a lipid surfactant and carried within theliposome's internal space; in these cases, the liposome membrane isformulated to resist the disruptive effects of the activeagent-surfactant aggregate.

According to one embodiment of the present invention, the lipid bilayerof a liposome contains lipids derivatized with polyethylene glycol(PEG), such that the PEG chains extend from the inner surface of thelipid bilayer into the interior space encapsulated by the liposome, andextend from the exterior of the lipid bilayer into the surroundingenvironment.

Active agents contained within liposomes of the present invention are insolubilized form. Aggregates of surfactant and active agent (such asemulsions or micelles containing the active agent of interest) may beentrapped within the interior space of liposomes according to thepresent invention. A surfactant acts to disperse and solubilize theactive agent, and may be selected from any suitable aliphatic,cycloaliphatic or aromatic surfactant, including but not limited tobiocompatible lysophosphatidylcholines (LPCs) of varying chain lengths(for example, from about C.sub.14 to about C.sub.20).Polymer-derivatized lipids such as PEG-lipids may also be utilized formicelle formation as they will act to inhibit micelle/membrane fusion,and as the addition of a polymer to surfactant molecules decreases theCMC of the surfactant and aids in micelle formation. Preferred aresurfactants with CMCs in the micromolar range; higher CMC surfactantsmay be utilized to prepare micelles entrapped within liposomes of thepresent invention, however, micelle surfactant monomers could affectliposome bilayer stability and would be a factor in designing a liposomeof a desired stability.

Liposomes according to the present invention may be prepared by any of avariety of techniques that are known in the art. See, e.g., U.S. Pat.No. 4,235,871; Published PCT applications WO 96/14057; New RRC,Liposomes: A practical approach, IRL Press, Oxford (1990), pages 33-104;Lasic DD, Liposomes from physics to applications, Elsevier SciencePublishers BV, Amsterdam, 1993.

For example, liposomes of the present invention may be prepared bydiffusing a lipid derivatized with a hydrophilic polymer into preformedliposomes, such as by exposing preformed liposomes to micelles composedof lipid-grafted polymers, at lipid concentrations corresponding to thefinal mole percent of derivatized lipid which is desired in theliposome. Liposomes containing a hydrophilic polymer can also be formedby homogenization, lipid-field hydration, or extrusion techniques, asare known in the art.

In one aspect of the present invention, the liposomes are prepared tohave substantially homogeneous sizes in a selected size range. Oneeffective sizing method involves extruding an aqueous suspension of theliposomes through a series of polycarbonate membranes having a selecteduniform pore size; the pore size of the membrane will correspond roughlywith the largest sizes of liposomes produced by extrusion through thatmembrane. See e.g., U.S. Pat. No. 4,737,323 (Apr. 12, 1988).

The release characteristics of a formulation of the present inventiondepend on the encapsulating material, the concentration of encapsulateddrug, and the presence of release modifiers. For example, release can bemanipulated to be pH dependent, for example, using a pH sensitivecoating that releases only at a low pH, as in the stomach, or a higherpH, as in the intestine. An enteric coating can be used to preventrelease from occurring until after passage through the stomach. Multiplecoatings or mixtures of cyanamide encapsulated in different materialscan be used to obtain an initial release in the stomach, followed bylater release in the intestine. Release can also be manipulated byinclusion of salts or pore forming agents, which can increase wateruptake or release of drug by diffusion from the capsule. Excipientswhich modify the solubility of the drug can also be used to control therelease rate. Agents which enhance degradation of the matrix or releasefrom the matrix can also be incorporated. They can be added to the drug,added as a separate phase (i.e., as particulates), or can beco-dissolved in the polymer phase depending on the compound. In allcases the amount should be between 0.1 and thirty percent (w/w polymer).Types of degradation enhancers include inorganic salts such as ammoniumsulfate and ammonium chloride, organic acids such as citric acid,benzoic acid, and ascorbic acid, inorganic bases such as sodiumcarbonate, potassium carbonate, calcium carbonate, zinc carbonate, andzinc hydroxide, and organic bases such as protamine sulfate, spermine,choline, ethanolamine, diethanolamine, and triethanolamine andsurfactants such as Tween® and Pluronic®. Pore forming agents which addmicrostructure to the matrices (i.e., water soluble compounds such asinorganic salts and sugars) are added as particulates. The range shouldbe between one and thirty percent (w/w polymer).

Uptake can also be manipulated by altering residence time of theparticles in the gut. This can be achieved, for example, by coating theparticle with, or selecting as the encapsulating material, a mucosaladhesive polymer. Examples include most polymers with free carboxylgroups, such as chitosan, celluloses, and especially polyacrylates (asused herein, polyacrylates refers to polymers including acrylate groupsand modified acrylate groups such as cyanoacrylates and methacrylates).

Pharmaceutical Combinations

The invention especially relates to the use of a compound of the formulaI (or a pharmaceutical composition comprising a compound of the formulaI) in the treatment of one or more of the diseases mentioned herein;wherein the response to treatment is beneficial as demonstrated, forexample, by the partial or complete removal of one or more of thesymptoms of the disease up to complete cure or remission.

A compound of formula (I) can also be used in combination with thefollowing compounds and antibody-drug conjugates:

BCR-ABL inhibitors: Imatinib (Gleevec®); Inilotinib hydrochloride;Nilotinib (Tasigna®); Dasatinib (BMS-345825); Bosutinib (SKI-606);Ponatinib (AP24534); Bafetinib (INNO406); Danusertib (PHA-739358),AT9283 (CAS 1133385-83-7); Saracatinib (AZD0530); andN-[2-[(1S,4R)-6-[[4-(Cyclobutylamino)-5-(trifluoromethyl)-2-pyrimidinyl]amino]-1,2,3,4-tetrahydronaphthalen-1,4-imin-9-yl]-2-oxoethyl]-acetamide(PF-03814735, CAS 942487-16-3); and LGX818.

ALK inhibitors: PF-2341066 (XALKORI®; crizotinib);5-chloro-N4-(2-(isopropylsulfonyl)phenyl)-N2-(2-methoxy-4-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)phenyl)pyrimidine-2,4-diamine;GSK1838705A; and CH5424802.

BRAF inhibitors: Vemurafanib (PLX4032); and Dabrafenib.

FLT3 inhibitors—sunitinib malate (sold under the tradename Sutent® byPfizer); PKC412 (midostaurin); tanutinib, sorafenib, sunitinib,midostaurin, lestaurtinib, KW-2449, quizartinib (AC220) and crenolanib.

MEK Inhibitors—trametinib.

Vascular Endothelial Growth Factor (VEGF) receptor inhibitors:Bevacizumab (sold under the trademark Avastin® by Genentech/Roche),axitinib,(N-methyl-2-[[3-[(E)-2-pyridin-2-ylethenyl]-1H-indazol-6-yl]sulfanyl]benzamide,also known as AG013736, and described in PCT Publication No. WO01/002369), Brivanib Alaninate((S)—((R)-1-(4-(4-Fluoro-2-methyl-1H-indol-5-yloxy)-5-methylpyrrolo[2,1-f][1,2,4]triazin-6-yloxy)propan-2-yl)2-aminopropanoate,also known as BMS-582664), motesanib(N-(2,3-dihydro-3,3-dimethyl-1H-indol-6-yl)-2-[(4-pyridinylmethyl)amino]-3-pyridinecarboxamide,and described in PCT Publication No. WO 02/066470), pasireotide (alsoknown as SOM230, and described in PCT Publication No. WO 02/010192),sorafenib (sold under the tradename Nexavar®);

HER2 receptor inhibitors: Trastuzumab (sold under the trademarkHerceptin® by Genentech/Roche), neratinib (also known as HKI-272,(2E)-N-[4-[[3-chloro-4-[(pyridin-2-yl)methoxy]phenyl]amino]-3-cyano-7-ethoxyquinolin-6-yl]-4-(dimethylamino)but-2-enamide,and described PCT Publication No. WO 05/028443), lapatinib or lapatinibditosylate (sold under the trademark Tykerb® by GlaxoSmithKline);Trastuzumab emtansine (in the United States, ado-trastuzumab emtansine,trade name Kadcyla)—an antibody-drug conjugate consisting of themonoclonal antibody trastuzumab (Herceptin) linked to the cytotoxicagent mertansine (DM1);

CD20 antibodies: Rituximab (sold under the trademarks Riuxan® andMabThera® by Genentech/Roche), tositumomab (sold under the trademarksBexxar® by GlaxoSmithKline), ofatumumab (sold under the trademarkArzerra® by GlaxoSmithKline);

Tyrosine kinase inhibitors: Erlotinib hydrochloride (sold under thetrademark Tarceva® by Genentech/Roche), Linifanib(N-[4-(3-amino-1H-indazol-4-yl)phenyl]-N′-(2-fluoro-5-methylphenyl)urea,also known as ABT 869, available from Genentech), sunitinib malate (soldunder the tradename Sutent® by Pfizer), bosutinib(4-[(2,4-dichloro-5-methoxyphenyl)amino]-6-methoxy-7-[3-(4-methylpiperazin-1-yl)propoxy]quinoline-3-carbonitrile,also known as SKI-606, and described in U.S. Pat. No. 6,780,996),dasatinib (sold under the tradename Sprycel® by Bristol-Myers Squibb),armala (also known as pazopanib, sold under the tradename Votrient® byGlaxoSmithKline), imatinib and imatinib mesylate (sold under thetradenames Gilvec® and Gleevec® by Novartis);

DNA Synthesis inhibitors: Capecitabine (sold under the trademark Xeloda®by Roche), gemcitabine hydrochloride (sold under the trademark Gemzar®by Eli Lilly and Company), nelarabine((2R,3S,4R,5R)-2-(2-amino-6-methoxy-purin-9-yl)-5-(hydroxymethyl)oxolane-3,4-diol,sold under the tradenames Arranon® and Atriance® by GlaxoSmithKline);

Antineoplastic agents: oxaliplatin (sold under the tradename Eloxatin®ay Sanofi-Aventis and described in U.S. Pat. No. 4,169,846);

Epidermal growth factor receptor (EGFR) inhibitors: Gefitnib (sold underthe tradename Iressa®),N-[4-[(3-Chloro-4-fluorophenyl)amino]-7-[[(3″S″)-tetrahydro-3-furanyl]oxy]-6-quinazolinyl]-4(dimethylamino)-2-butenamide,sold under the tradename Tovok® by Boehringer Ingelheim), cetuximab(sold under the tradename Erbitux® by Bristol-Myers Squibb), panitumumab(sold under the tradename Vectibix® by Amgen);

HER dimerization inhibitors: Pertuzumab (sold under the trademarkOmnitarg®, by Genentech);

Human Granulocyte colony-stimulating factor (G-CSF) modulators:Filgrastim (sold under the tradename Neupogen® by Amgen);

Immunomodulators: Afutuzumab (available from Roche®), pegfilgrastim(sold under the tradename Neulasta® by Amgen), lenalidomide (also knownas CC-5013, sold under the tradename Revlimid®), thalidomide (sold underthe tradename Thalomid®);

CD40 inhibitors: Dacetuzumab (also known as SGN-40 or huS2C6, availablefrom Seattle Genetics, Inc);

Pro-apoptotic receptor agonists (PARAs): Dulanermin (also known asAMG-951, available from Amgen/Genentech);

Hedgehog antagonists:2-chloro-N-[4-chloro-3-(2-pyridinyl)phenyl]-4-(methylsulfonyl)-benzamide(also known as GDC-0449, and described in PCT Publication No. WO06/028958);

PI3K inhibitors:4-[2-(1H-Indazol-4-yl)-6-[[4-(methylsulfonyl)piperazin-1-yl]methyl]thieno[3,2-d]pyrimidin-4-yl]morpholine(also known as GDC 0941 and described in PCT Publication Nos. WO09/036082 and WO 09/055730),2-Methyl-2-[4-[3-methyl-2-oxo-8-(quinolin-3-yl)-2,3-dihydroimidazo[4,5-c]quinolin-1-yl]phenyl]propionitrile(also known as BEZ 235 or NVP-BEZ 235, and described in PCT PublicationNo. WO 06/122806);

Phospholipase A2 inhibitors: Anagrelide (sold under the tradenameAgrylin®);

BCL-2 inhibitors:4-[4-[[2-(4-chlorophenyl)-5,5-dimethyl-1-cyclohexen-1-yl]methyl]-1-piperazinyl]-N-[[4-[[(1R)-3-(4-morpholinyl)-1-[(phenylthio)methyl]propyl]amino]-3-[(trifluoromethyl)sulfonyl]phenyl]sulfonyl]benzamide(also known as ABT-263 and described in PCT Publication No. WO09/155386);

Mitogen-activated protein kinase kinase (MEK) inhibitors: XL-518 (CasNo. 1029872-29-4, available from ACC Corp.);

Aromatase inhibitors: Exemestane (sold under the trademark Aromasin® byPfizer), letrozole (sold under the tradename Femara® by Novartis),anastrozole (sold under the tradename Arimidex®);

Topoisomerase I inhibitors: Irinotecan (sold under the trademarkCamptosar® by Pfizer), topotecan hydrochloride (sold under the tradenameHycamtin® by GlaxoSmithKline);

Topoisomerase II inhibitors: etoposide (also known as VP-16 andEtoposide phosphate, sold under the tradenames Toposar®, VePesid® andEtopophos®), teniposide (also known as VM-26, sold under the tradenameVumon®);

mTOR inhibitors: Temsirolimus (sold under the tradename Torisel® byPfizer), ridaforolimus (formally known as deferolimus,(1R,2R,4S)-4-[(2R)-2[(1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28Z,30S,32S,35R)-1,18-dihydroxy-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-2,3,10,14,20-pentaoxo-11,36-dioxa-4-azatricyclo[30.3.1.0^(4.9)]hexatriaconta-16,24,26,28-tetraen-12-yl]propyl]-2-methoxycyclohexyldimethylphosphinate, also known as AP23573 and MK8669, and described inPCT Publication No. WO 03/064383), everolimus (sold under the tradenameAfinitor® by Novartis);

Osteoclastic bone resorption inhibitors:1-Hydroxy-2-imidazol-1-yl-phosphonoethyl) phosphonic acid monohydrate(sold under the tradename Zometa® by Novartis);

CD33 Antibody Drug Conjugates: Gemtuzumab ozogamicin (sold under thetradename Mylotarg® by Pfizer/Wyeth);

CD22 Antibody Drug Conjugates: Inotuzumab ozogamicin (also referred toas CMC-544 and WAY-207294, available from Hangzhou Sage Chemical Co.,Ltd.);

CD20 Antibody Drug Conjugates: Ibritumomab tiuxetan (sold under thetradename Zevalin®);

Somatostain analogs: octreotide (also known as octreotide acetate, soldunder the tradenames Sandostatin® and Sandostatin LAR®);

Synthetic Interleukin-11 (IL-11): oprelvekin (sold under the tradenameNeumega® by Pfizer/Wyeth);

Synthetic erythropoietin: Darbepoetin alfa (sold under the tradenameAranesp® by Amgen);

Receptor Activator for Nuclear Factor κ B (RANK) inhibitors: Denosumab(sold under the tradename Prolia® by Amgen);

Thrombopoietin mimetic peptibodies: Romiplostim (sold under thetradename Nplate® by Amgen;

Cell growth stimulators: Palifermin (sold under the tradename Kepivance®by Amgen);

Anti-Insulin-like Growth Factor-1 receptor (IGF-1R) antibodies:Figitumumab (also known as CP-751,871, available from ACC Corp),robatumumab (CAS No. 934235-44-6);

Anti-CS1 antibodies: Elotuzumab (HuLuc63, CAS No. 915296-00-3);

CD52 antibodies: Alemtuzumab (sold under the tradename Campath®);

CTLA-4 inhibitors: Tremelimumab (IgG2 monoclonal antibody available fromPfizer, formerly known as ticilimumab, CP-675,206), ipilimumab (CTLA-4antibody, also known as MDX-010, CAS No. 477202-00-9);

PD1 inhibitors: Nivolumab (also referred to herein as MDX-1106,MDX-1106-04, ONO-4538, BMS0936558, CAS Registry No: 946414-94-4)disclosed in, for example, U.S. Pat. No. 8,008,449, and having asequence disclosed therein (or a sequence substantially identical orsimilar thereto, for example, a sequence having at least 85%, 90%, 95%identity or greater to the sequence specified in U.S. Pat. No.8,008,449); Pembrolizumab (also referred to herein as Lambrolizumab,MK-3475, MK03475, SCH-900475 or KEYTRUDA), disclosed in, for example,U.S. Pat. No. 8,354,509 and WO 2009/114335, and having a sequencedisclosed therein (or a sequence substantially identical or similarthereto, for example, a sequence having at least 85%, 90%, 95% identityor greater to the sequence specified in U.S. Pat. No. 8,354,509 andWO2009/114335); an immunoadhesin (for example, an immunoadhesincomprising an extracellular or PD-1 binding portion of PD-L1 or PD-L2fused to a constant region (e.g., an Fc region of an immunoglobulinsequence); Pidilizumab (CT-011; Cure Tech) is a humanized IgG1kmonoclonal antibody that binds to PD1 (Pidilizumab and other humanizedanti-PD-1 monoclonal antibodies are disclosed in WO2009/101611); andAMP-224 (B7-DCIg; Amplimmune), disclosed in WO2010/027827 andWO2011/066342), is a PD-L2 Fc fusion soluble receptor that blocks theinteraction between PD1 and B7-H1; other PD-1 inhibitors, for example,anti-PD1 antibodies disclosed in U.S. Pat. No. 8,609,089, US 2010028330,and/or US 20120114649.

PDL1 inhibitors: MSB0010718C (also referred to as A09-246-2; MerckSerono) is a monoclonal antibody that binds to PD-L1 and is disclosedin, for example, WO 2013/0179174, (and having a sequence substantiallyidentical or similar thereto, for example, a sequence having at least85%, 90%, 95% identity or higher to the sequence specified in WO2013/0179174); and anti-PD-L1 binding antagonist selected fromYW243.55.570, MPDL3280A (Genetech/Roche) is a human Fc optimized IgG1monoclonal antibody that binds to PD-L1 (MDPL3280A and other humanmonoclonal antibodies to PD-L1 are disclosed in U.S. Pat. No. 7,943,743and U.S Publication No.: 20120039906); MEDI-4736, MSB-0010718C, orMDX-1105 (MDX-1105, also known as BMS-936559, is an anti-PD-L1 antibodydescribed in WO2007/005874; antibody YW243.55.S70 is an anti-PD-L1described in WO 2010/077634).

LAG-3 inhibitors: BMS-986016 (also referred to as BMS986016;Bristol-Myers Squibb) is a monoclonal antibody that binds to LAG-3.BMS-986016 and other humanized anti-LAG-3 antibodies are disclosed in US2011/0150892, WO2010/019570, and WO2014/008218.

GITR agonists: exemplary GITR agonists include, for example., GITRfusion proteins and anti-GITR antibodies (for example, bivalentanti-GITR antibodies), such as, a GITR fusion protein described in U.S.Pat. No. 6,111,090, European Patent No.: 090505B1, U.S. Pat. No.8,586,023, PCT Publication Nos.: WO 2010/003118 and 2011/090754, or ananti-GITR antibody described, for example, in U.S. Pat. No. 7,025,962,European Patent No.: 1947183B1, U.S. Pat. Nos. 7,812,135, 8,388,967,8,591,886, European Patent No.: EP 1866339, PCT Publication No.: WO2011/028683, PCT Publication No.:WO 2013/039954, PCT Publication No.:WO2005/007190, PCT Publication No.: WO 2007/133822, PCT Publication No.:WO2005/055808, PCT Publication No.: WO 99/40196, PCT Publication No.: WO2001/03720, PCT Publication No.: WO99/20758, PCT Publication No.:WO2006/083289, PCT Publication No.: WO 2005/115451, U.S. Pat. No.7,618,632, and PCT Publication No.: WO 2011/051726.

Histone deacetylase inhibitors (HDI): Voninostat (sold under thetradename Zolinza® by Merck).

anti-CTLA4 antibodies include Tremelimumab (IgG2 monoclonal antibodyavailable from Pfizer, formerly known as ticilimumab, CP-675,206); andIpilimumab (CTLA-4 antibody, also known as MDX-010, CAS No.477202-00-9).

anti-TIM-3 antibody or antigen-binding fragment thereof.

Alkylating agents: Temozolomide (sold under the tradenames Temodar® andTemodal® by Schering-Plough/Merck), dactinomycin (also known asactinomycin-D and sold under the tradename Cosmegen®), melphalan (alsoknown as L-PAM, L-sarcolysin, and phenylalanine mustard, sold under thetradename Alkeran®), altretamine (also known as hexamethylmelamine(HMM), sold under the tradename Hexalen®), carmustine (sold under thetradename BiCNU®), bendamustine (sold under the tradename Treanda®),busulfan (sold under the tradenames Busulfex® and Myleran®), carboplatin(sold under the tradename Paraplatin®), lomustine (also known as CCNU,sold under the tradename CeeNU®), cisplatin (also known as CDDP, soldunder the tradenames Platinol® and Platinol®-AQ), chlorambucil (soldunder the tradename Leukeran®), cyclophosphamide (sold under thetradenames Cytoxan® and Neosar®), dacarbazine (also known as DTIC, DICand imidazole carboxamide, sold under the tradename DTIC-Dome®),altretamine (also known as hexamethylmelamine (HMM) sold under thetradename Hexalen®), ifosfamide (sold under the tradename Ifex®),procarbazine (sold under the tradename Matulane®), mechlorethamine (alsoknown as nitrogen mustard, mustine and mechloroethamine hydrochloride,sold under the tradename Mustargen®), streptozocin (sold under thetradename Zanosar®), thiotepa (also known as thiophosphoamide, TESPA andTSPA, sold under the tradename Thioplex®;

Biologic response modifiers: bacillus calmette-guerin (sold under thetradenames theraCys® and TICE@ BCG), denileukin diftitox (sold under thetradename Ontak®);

Anti-tumor antibiotics: doxorubicin (sold under the tradenamesAdriamycin® and Rubex®), bleomycin (sold under the tradename Lenoxane®),daunorubicin (also known as dauorubicin hydrochloride, daunomycin, andrubidomycin hydrochloride, sold under the tradename Cerubidine®),daunorubicin liposomal (daunorubicin citrate liposome, sold under thetradename DaunoXome®), mitoxantrone (also known as DHAD, sold under thetradename Novantrone®), epirubicin (sold under the tradename Ellence™),idarubicin (sold under the tradenames Idamycin®, Idamycin PFS®),mitomycin C (sold under the tradename Mutamycin®);

Anti-microtubule agents: Estramustine (sold under the tradename Emcyl®);

Cathepsin K inhibitors: Odanacatib (also know as MK-0822,N-(1-cyanocyclopropyl)-4-fluoro-N²-{(1S)-2,2,2-trifluoro-1-[4′-(methylsulfonyl)biphenyl-4-yl]ethyl}-L-leucinamide,available from Lanzhou Chon Chemicals, ACC Corp., and ChemieTek, anddescribed in PCT Publication no. WO 03/075836);

Epothilone B analogs: Ixabepilone (sold under the tradename Lxempra® byBristol-Myers Squibb);

Heat Shock Protein (HSP) inhibitors: Tanespimycin(17-allylamino-17-demethoxygeldanamycin, also known as KOS-953 and17-AAG, available from SIGMA, and described in U.S. Pat. No. 4,261,989);

TpoR agonists: Eltrombopag (sold under the tradenames Promacta® andRevolade® by GlaxoSmithKline);

Anti-mitotic agents: Docetaxel (sold under the tradename Taxotere® bySanofi-Aventis);

Adrenal steroid inhibitors: aminoglutethimide (sold under the tradenameCytadren®);

Anti-androgens: Nilutamide (sold under the tradenames Nilandron® andAnandron®), bicalutamide (sold under tradename Casodex®), flutamide(sold under the tradename Fulexin™);

Androgens: Fluoxymesterone (sold under the tradename Halotestin®);

Proteasome inhibitors: Bortezomib (sold under the tradename Velcade®);

CDK1 inhibitors: Alvocidib (also known as flovopirdol or HMR-1275,2-(2-chlorophenyl)-5,7-dihydroxy-8-[(3S,4R)-3-hydroxy-1-methyl-4-piperidinyl]-4-chromenone,and described in U.S. Pat. No. 5,621,002);

Gonadotropin-releasing hormone (GnRH) receptor agonists: Leuprolide orleuprolide acetate (sold under the tradenames Viadure® by Bayer AG,Eligard® by Sanofi-Aventis and Lupron® by Abbott Lab);

Taxane anti-neoplastic agents: Cabazitaxel(1-hydroxy-7β,10β-dimethoxy-9-oxo-5β,20-epoxytax-11-ene-2α,4,13α-triyl-4-acetate-2-benzoate-13-[(2R,3S)-3-{[(tert-butoxy)carbonyl]amino}-2-hydroxy-3-phenylpropanoate),larotaxel((2α,3ξ,4α,5β,7α,10β,13α)-4,10-bis(acetyloxy)-13-({(2R,3S)-3-[(tert-butoxycarbonyl)amino]-2-hydroxy-3-phenylpropanoyl}oxy)-1-hydroxy-9-oxo-5,20-epoxy-7,19-cyclotax-11-en-2-ylbenzoate);

5HT1a receptor agonists: Xaliproden (also known as SR57746,1-[2-(2-naphthyl)ethyl]-4-[3-(trifluoromethyl)phenyl]-1,2,3,6-tetrahydropyridine,and described in U.S. Pat. No. 5,266,573);

HPC vaccines: Cervarix® sold by GlaxoSmithKline, Gardasil® sold byMerck;

Iron Chelating agents: Deferasinox (sold under the tradename Exjade® byNovartis);

Anti-metabolites: Claribine (2-chlorodeoxyadenosine, sold under thetradename Leustatin®), 5-fluorouracil (sold under the tradenameAdrucil®), 6-thioguanine (sold under the tradename Purinethol®),pemetrexed (sold under the tradename Alimta®), cytarabine (also known asarabinosylcytosine (Ara-C), sold under the tradename Cytosar-U®),cytarabine liposomal (also known as Liposomal Ara-C, sold under thetradename DepoCyt™), decitabine (sold under the tradename Dacogen®),hydroxyurea (sold under the tradenames Hydrea®, Droxia™ and Mylocel™),fludarabine (sold under the tradename Fludara®), floxuridine (sold underthe tradename FUDR®), cladribine (also known as 2-chlorodeoxyadenosine(2-CdA) sold under the tradename Leustatin™), methotrexate (also knownas amethopterin, methotrexate sodim (MTX), sold under the tradenamesRheumatrex® and Trexall™), pentostatin (sold under the tradenameNipent®);

Bisphosphonates: Pamidronate (sold under the tradename Aredia®),zoledronic acid (sold under the tradename Zometa®);

Demethylating agents: 5-azacitidine (sold under the tradename Vidaza®),decitabine (sold under the tradename Dacogen®);

Plant Alkaloids: Paclitaxel protein-bound (sold under the tradenameAbraxane®), vinblastine (also known as vinblastine sulfate,vincaleukoblastine and VLB, sold under the tradenames Alkaban-AQ® andVelban®), vincristine (also known as vincristine sulfate, LCR, and VCR,sold under the tradenames Oncovin® and Vincasar Pfs®), vinorelbine (soldunder the tradename Navelbine®), paclitaxel (sold under the tradenamesTaxol and Onxal™);

Retinoids: Alitretinoin (sold under the tradename Panretin®), tretinoin(all-trans retinoic acid, also known as ATRA, sold under the tradenameVesanoid®), Isotretinoin (13-cis-retinoic acid, sold under thetradenames Accutane®, Amnesteem®, Claravis®, Clarus®, Decutan®,Isotane®, Izotech®, Oratane®, Isotret®, and Sotret®), bexarotene (soldunder the tradename Targretin®);

Glucocorticosteroids: Hydrocortisone (also known as cortisone,hydrocortisone sodium succinate, hydrocortisone sodium phosphate, andsold under the tradenames Ala-Cort®, Hydrocortisone Phosphate,Solu-Cortef®, Hydrocort Acetate® and Lanacort®), dexamethazone((8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-17-(2-hydroxyacetyl)-10,13,16-trimethyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-3-one),prednisolone (sold under the tradenames Delta-Cortel®, Orapred®,Pediapred® and Prelone®), prednisone (sold under the tradenamesDeltasone®, Liquid Red®, Meticorten® and Orasone®), methylprednisolone(also known as 6-Methylprednisolone, Methylprednisolone Acetate,Methylprednisolone Sodium Succinate, sold under the tradenamesDuralone®, Medralone®, Medrol®, M-Prednisol® and Solu-Medrol®);

Cytokines: interleukin-2 (also known as aldesleukin and IL-2, sold underthe tradename Proleukin®), interleukin-11 (also known as oprevelkin,sold under the tradename Neumega®), alpha interferon alfa (also known asIFN-alpha, sold under the tradenames Intron® A, and Roferon-A®);

Estrogen receptor downregulators: Fulvestrant (sold under the tradenameFaslodex®);

Anti-estrogens: tamoxifen (sold under the tradename Novaldex®);

Toremifene (sold under the tradename Fareston®);

Selective estrogen receptor modulators (SERMs): Raloxifene (sold underthe tradename Evista®);

Leutinizing hormone releasing hormone (LHRH) agonists: Goserelin (soldunder the tradename Zoladex®);

Progesterones: megestrol (also known as megestrol acetate, sold underthe tradename Megace®);

Miscellaneous cytotoxic agents: Arsenic trioxide (sold under thetradename Trisenox®), asparaginase (also known as L-asparaginase,Erwinia L-asparaginase, sold under the tradenames Elspar® andKidrolase®);

A compound of formula (I) can also be used in combination with thefollowing adjunct therapies:

Anti-nausea drugs: NK-1 receptor antagonists: Casopitant (sold under thetradenames Rezonic® and Zunrisa® by GlaxoSmithKline); and

Cytoprotective agents: Amifostine (sold under the tradename Ethyol®),leucovorin (also known as calcium leucovorin, citrovorum factor andfolinic acid).

Immune checkpoint inhibitors: In one embodiment, the combinationtherapies disclosed herein include an inhibitor of an inhibitorymolecule of an immune checkpoint molecule. The term “immune checkpoints”refers to a group of molecules on the cell surface of CD4 and CD8 Tcells. These molecules can effectively serve as “brakes” todown-modulate or inhibit an anti-tumor immune response. Immunecheckpoint molecules include, but are not limited to, Programmed Death 1(PD-1), Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4), B7H1, B7H4, OX-40,CD137, CD40, and LAG3, which directly inhibit immune cells,immunotherapeutic agents which can act as immune checkpoint inhibitorsuseful in the methods of the present invention, include, but are notlimited to, inhibitors of PD-L1, PD-L2, CTLA4, TIM3, LAG3, VISTA, BTLA,TIGIT, LAIRI, CD160, 2B4 and/or TGFR beta. Inhibition of an inhibitorymolecule can be performed by inhibition at the DNA, RNA or proteinlevel. In embodiments, an inhibitory nucleic acid (e.g., a dsRNA, siRNAor shRNA), can be used to inhibit expression of an inhibitory molecule.In other embodiments, the inhibitor of an inhibitory signal is, apolypeptide e.g., a soluble ligand, or an antibody or antigen-bindingfragment thereof, that binds to the inhibitory molecule.

In certain embodiments, the anti-PD-1 molecules described herein areadministered in combination with one or more other inhibitors of PD-1,PD-L1 and/or PD-L2 known in the art. The antagonist may be an antibody,an antigen binding fragment thereof, an immunoadhesin, a fusion protein,or oligopeptide.

In certain embodiments, the combination therapies disclosed hereininclude a modulator of a costimulatory molecule or an inhibitorymolecule, e.g., a co-inhibitory ligand or receptor.

In one embodiment, the costimulatory modulator, e.g., agonist, of acostimulatory molecule is chosen from an agonist (e.g., an agonisticantibody or antigen-binding fragment thereof, or soluble fusion) ofOX40, CD2, CD27, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), 4-1BB(CD137), GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7,NKp80, CD160, B7-H3 or CD83 ligand.

In another embodiment, the combination therapies disclosed hereininclude a costimulatory molecule, e.g., an agonist associated with apositive signal that includes a costimulatory domain of CD28, CD27, ICOSand GITR.

In one embodiment, the anti-PD-1 antibody molecule is administered aftertreatment, e.g., after treatment of a melanoma, with an anti-CTLA4antibody (e.g., ipilimumab) with or without a BRAF inhibitor (e.g.,vemurafenib or dabrafenib). Exemplary doses that can be use include adose of anti-PD-1 antibody molecule of about 1 to 10 mg/kg, e.g., 3mg/kg, and a dose of an anti-CTLA-4 antibody, e.g., ipilimumab, of about3 mg/kg.

In another embodiment, the anti-PD-1 or PD-L1 antibody molecule isadministered in combination with an anti-LAG-3 antibody or anantigen-binding fragment thereof. In another embodiment, the anti-PD-1or PD-L1 antibody molecule is administered in combination with ananti-TIM-3 antibody or antigen-binding fragment thereof. In yet otherembodiments, the anti-PD-1 or PD-L1 antibody molecule is administered incombination with an anti-LAG-3 antibody and an anti-TIM-3 antibody, orantigen-binding fragments thereof. The combination of antibodies recitedherein can be administered separately, e.g., as separate antibodies, orlinked, e.g., as a bispecific or trispecific antibody molecule. In oneembodiment, a bispecific antibody that includes an anti-PD-1 or PD-L1antibody molecule and an anti-TIM-3 or anti-LAG-3 antibody, orantigen-binding fragment thereof, is administered. In certainembodiments, the combination of antibodies recited herein is used totreat a cancer, e.g., a cancer as described herein (e.g., a solidtumor). The efficacy of the aforesaid combinations can be tested inanimal models known in the art. For example, the animal models to testthe synergistic effect of anti-PD-1 and anti-LAG-3 are described, e.g.,in Woo et al. (2012) Cancer Res. 72(4):917-27).

In some embodiments, the combination therapy disclosed herein (e.g., ananti-PD-1 or PD-L1 antibody molecule, alone or in combination withanother immunomodulator (e.g., an anti-LAG-3, or anti-TIM-3 antibodymolecule). In one embodiment, the anti-PD-1 or PD-L1 antibody moleculeis administered in combination with an anti-LAG-3 antibody or anantigen-binding fragment thereof. In another embodiment, the anti-PD-1or PD-L 1 antibody molecule is administered in combination with ananti-TIM-3 antibody or antigen-binding fragment thereof. In yet otherembodiments, the anti-PD-1 or PD-L1 antibody molecule is administered incombination with an anti-LAG-3 antibody and an anti-TIM-3 antibody, orantigen-binding fragments thereof. The combination of antibodies recitedherein can be administered separately, e.g., as separate antibodies, orlinked, e.g., as a bispecific or trispecific antibody molecule. In oneembodiment, a bispecific antibody that includes an anti-PD-1 or PD-L1antibody molecule and an anti-TIM-3 or anti-LAG-3 antibody, orantigen-binding fragment thereof, is administered. In certainembodiments, the combination of antibodies recited herein is used totreat a cancer, e.g., a cancer as described herein (e.g., a solidtumor). The efficacy of the aforesaid combinations can be tested inanimal models known in the art. For example, the animal models to testthe synergistic effect of anti-PD-1 and anti-LAG-3 are described, e.g.,in Woo et al. (2012) Cancer Res. 72(4):917-27).24

In certain embodiments, the antibody molecule is in the form of abispecific or multispecific antibody molecule. In one embodiment, thebispecific antibody molecule has a first binding specificity to PD-1 orPD-L1 and a second binding specifity, e.g., a second binding specificityto TIM-3, LAG-3, or PD-L2. In one embodiment, the bispecific antibodymolecule binds to PD-1 or PD-L 1 and TIM-3. In another embodiment, thebispecific antibody molecule binds to PD-1 or PD-L1 and LAG-3. Inanother embodiment, the bispecific antibody molecule binds to PD-1 orPD-L 1. In yet another embodiment, the bispecific antibody moleculebinds to PD-1 and PD-L2. In another embodiment, the bispecific antibodymolecule binds to TIM-3 and LAG-3. Any combination of the aforesaidmolecules can be made in a multispecific antibody molecule, e.g., atrispecific antibody that includes a first binding specificity to PD-1or PD-1, and a second and third binding specifities to two or more of:TIM-3, LAG-3, or PD-L2.

None of the quotations of references made within the present disclosureis to be understood as an admission that the references cited are priorart that would negatively affect the patentability of the presentinvention.

Processes for Making Compounds of the Invention

The present invention also includes processes for the preparation ofcompounds of the invention. In the reactions described, it can benecessary to protect reactive functional groups, for example hydroxy,amino, imino, thio or carboxy groups, where these are desired in thefinal product, to avoid their unwanted participation in the reactions.Conventional protecting groups can be used in accordance with standardpractice, for example, see T. W. Greene and P. G. M. Wuts in “ProtectiveGroups in Organic Chemistry”, John Wiley and Sons, 1991.

Compounds of Formula I can be prepared by proceeding as in the followingReaction Scheme I:

in which p, q, Y₁, Y₂, R_(2a), R_(2b), R_(3a), R_(3b), R_(4a), R_(4b),R_(5a), R_(5b), R₇ and R₈ are as defined for Formula I in the Summary ofthe Invention, Q is a halogen (like bromine) or a thiol, boronate orstannate which reacts with a halogen on compound 5, and X is a reactivegroup which reacts with Q (such as a boronate, stannane, alcohol, thiol,halogen, and the like). Compound 4 may be prepared by reacting compound2 with compound 3 through a reaction under suitable acid or baseconditions in the presence or absence of a transition metal underambient temperature, or under thermal or microwave conditions.Alternatively, the halogen of compound 2 may be replaced by otherhalogens or suitable activating groups such as triflates, mesylates,tosylates, nonaflates, boronates, organostannanes, organosilyls,organozincs, lithium, magnesium, and the like.

A compound of formula I can be prepared by reacting compound 4 with asuitable coupling partner (e.g. compound 5) depending on X. For example,compound 5 is shown in reaction scheme I as a substituted phenyl grouplinked via X. Alternatively, compound 5 could be aryl-alcohol,aryl-thio, aryl-boronate, aryl-stannate, heteroaryl-alcohol, aryl-thiol,heteroaryl-thiol, aryl-boronate, aryl-stannane, olefin, or otheraryl-metals or heteroaryl-metals, and the like. The coupling partnersmay also be substituted. This reaction may be conducted under suitableacid or base conditions, in the presence or absence of a transitionmetal such as palladium, under ambient temperature, or under thermal ormicrowave conditions. Other halogens or suitable activating groups(e.g., triflates, mesylates, tosylates, and nonaflates) may be used inplace of Br for these transformations.

Alternatively, the coupling partners could be reversed and compound 2may be derivatized to a stannane, boronate, organo-zinc, organo-lithium,organo-magnesium, organo-silicon, organo-cuprate and coupled with asuitable aryl-halide, heteroaryl-halide, olefin or suitable reactivefunctional group (e.g., triflates, mesylates, tosylates and nonaflates),and the like.

These reactions may be conducted in the order described or in reverseorder, under a variety of solvents, temperatures, pressures, and undersuitable atmospheres. The reactions may be conducted under acid, base,and or transition metal conditions.

Detailed examples of the synthesis of compounds of Formula I can befound in the Examples, infra.

Additional Processes for Making Compounds of the Invention

A compound of the invention can be prepared as a pharmaceuticallyacceptable acid addition salt by reacting the free base form of thecompound with a pharmaceutically acceptable inorganic or organic acid.Alternatively, a pharmaceutically acceptable base addition salt of acompound of the invention can be prepared by reacting the free acid formof the compound with a pharmaceutically acceptable inorganic or organicbase.

Compounds of the formula I can also be modified by appending appropriatefunctionalities to enhance selective biological properties.Modifications of this kind are known in the art and include those thatincrease penetration into a given biological system (e.g. blood,lymphatic system, central nervous system, testis), increasebioavailability, increase solubility to allow parenteral administration(e.g. injection, infusion), alter metabolism and/or alter the rate ofsecretion. Examples of this type of modifications include but are notlimited to esterification, e.g. with polyethylene glycols,derivatisation with pivaloyloxy or fatty acid substituents, conversionto carbamates, hydroxylation of aromatic rings and heteroatomsubstitution in aromatic rings. Whereever compounds of the formula I,and/or N-oxides, tautomers and/or (preferably pharmaceuticallyacceptable) salts thereof are mentioned, this comprises such modifiedformulae, while preferably the molecules of the formula I, theirN-oxides, their tautomers and/or their salts are meant.

Alternatively, the salt forms of the compounds of the invention can beprepared using salts of the starting materials or intermediates. In viewof the close relationship between the novel compounds of the formula Iin free form and those in the form of their salts, including those saltsthat can be used as intermediates, for example in the purification oridentification of the novel compounds, any reference to the compounds ora compound of the formula I hereinbefore and hereinafter is to beunderstood as referring to the compound in free form and/or also to oneor more salts thereof, as appropriate and expedient, as well as to oneor more solvates, e.g. hydrates.

Salts are formed, for example, as acid addition salts, preferably withorganic or inorganic acids, from compounds of formula I with a basicnitrogen atom, especially the pharmaceutically acceptable salts.Suitable inorganic acids are, for example, halogen acids, such ashydrochloric acid, sulfuric acid, or phosphoric acid. Suitable organicacids are, for example, carboxylic, phosphonic, sulfonic or sulfamicacids, for example acetic acid, propionic acid, octanoic acid, decanoicacid, dodecanoic acid, glycolic acid, lactic acid, fumaric acid,succinic acid, malonic acid, adipic acid, pimelic acid, suberic acid,azelaic acid, malic acid, tartaric acid, citric acid, amino acids, suchas glutamic acid or aspartic acid, maleic acid, hydroxymaleic acid,methylmaleic acid, cyclohexanecarboxylic acid, adamantanecarboxylicacid, benzoic acid, salicylic acid, 4-aminosalicylic acid, phthalicacid, phenylacetic acid, mandelic acid, cinnamic acid, methane- orethane-sulfonic acid, 2-hydroxyethanesulfonic acid,ethane-1,2-disulfonic acid, benzenesulfonic acid, 4-toluenesulfonicacid, 2-naphthalenesulfonic acid, 1,5-naphthalene-disulfonic acid, 2- or3-methylbenzenesulfonic acid, methylsulfuric acid, ethylsulfuric acid,dodecylsulfuric acid, N-cyclohexylsulfamic acid, N-methyl-, N-ethyl- orN-propyl-sulfamic acid, or other organic protonic acids, such asascorbic acid.

For isolation or purification purposes it is also possible to usepharmaceutically unacceptable salts, for example picrates orperchlorates. For therapeutic use, only pharmaceutically acceptablesalts or free compounds are employed (where applicable in the form ofpharmaceutical preparations), and these are therefore preferred.

The free acid or free base forms of the compounds of the invention canbe prepared from the corresponding base addition salt or acid additionsalt from, respectively. For example a compound of the invention in anacid addition salt form can be converted to the corresponding free baseby treating with a suitable base (e.g., ammonium hydroxide solution,sodium hydroxide, and the like). A compound of the invention in a baseaddition salt form can be converted to the corresponding free acid bytreating with a suitable acid (e.g., hydrochloric acid, etc.).

Compounds of the invention in unoxidized form can be prepared fromN-oxides of compounds of the invention by treating with a reducing agent(e.g., sulfur, sulfur dioxide, triphenyl phosphine, lithium borohydride,sodium borohydride, phosphorus trichloride, tribromide, or the like) ina suitable inert organic solvent (e.g. acetonitrile, ethanol, aqueousdioxane, or the like) at 0 to 80° C.

Prodrug derivatives of the compounds of the invention can be prepared bymethods known to those of ordinary skill in the art (e.g., for furtherdetails see Saulnier et al., (1994), Bioorganic and Medicinal ChemistryLetters, Vol. 4, p. 1985). For example, appropriate prodrugs can beprepared by reacting a non-derivatized compound of the invention with asuitable carbamylating agent (e.g., 1,1-acyloxyalkylcarbanochloridate,para-nitrophenyl carbonate, or the like).

Protected derivatives of the compounds of the invention can be made bymeans known to those of ordinary skill in the art. A detaileddescription of techniques applicable to the creation of protectinggroups and their removal can be found in T. W. Greene, “ProtectingGroups in Organic Chemistry”, 3^(rd) edition, John Wiley and Sons, Inc.,1999.

Compounds of the present invention can be conveniently prepared, orformed during the process of the invention, as solvates (e.g.,hydrates). Hydrates of compounds of the present invention can beconveniently prepared by recrystallization from an aqueous/organicsolvent mixture, using organic solvents such as dioxin, tetrahydrofuranor methanol.

Compounds of the invention can be prepared as their individualstereoisomers by reacting a racemic mixture of the compound with anoptically active resolving agent to form a pair of diastereoisomericcompounds, separating the diastereomers and recovering the opticallypure enantiomers. While resolution of enantiomers can be carried outusing covalent diastereomeric derivatives of the compounds of theinvention, dissociable complexes are preferred (e.g., crystallinediastereomeric salts). Diastereomers have distinct physical properties(e.g., melting points, boiling points, solubilities, reactivity, etc.)and can be readily separated by taking advantage of thesedissimilarities. The diastereomers can be separated by chromatography,or preferably, by separation/resolution techniques based upondifferences in solubility. The optically pure enantiomer is thenrecovered, along with the resolving agent, by any practical means thatwould not result in racemization. A more detailed description of thetechniques applicable to the resolution of stereoisomers of compoundsfrom their racemic mixture can be found in Jean Jacques, Andre Collet,Samuel H. Wilen, “Enantiomers, Racemates and Resolutions”, John WileyAnd Sons, Inc., 1981.

In summary, the compounds of Formula I can be made by a process, whichinvolves:

(a) that of reaction scheme I; and

(b) optionally converting a compound of the invention into apharmaceutically acceptable salt;

(c) optionally converting a salt form of a compound of the invention toa non-salt form;

(d) optionally converting an unoxidized form of a compound of theinvention into a pharmaceutically acceptable N-oxide;

(e) optionally converting an N-oxide form of a compound of the inventionto its unoxidized form;

(f) optionally resolving an individual isomer of a compound of theinvention from a mixture of isomers;

(g) optionally converting a non-derivatized compound of the inventioninto a pharmaceutically acceptable prodrug derivative; and

(h) optionally converting a prodrug derivative of a compound of theinvention to its non-derivatized form.

Insofar as the production of the starting materials is not particularlydescribed, the compounds are known or can be prepared analogously tomethods known in the art or as disclosed in the Examples hereinafter.

One of skill in the art will appreciate that the above transformationsare only representative of methods for preparation of the compounds ofthe present invention, and that other well known methods can similarlybe used.

EXAMPLES

The following examples and intermediates serve to illustrate theinvention without limiting the scope thereof. Some abbreviations used inthe examples are as follows: acetic acid (AcOH); triethylamine (TEA);tetrahydrofuran (THF); aqueous (aq.); atmosphere (atm.);2,2′-bis-diphenylphosphanyl-[1,1′]binaphthalenyl (BINAP);4-dimethylaminopyridine (DMAP); tert-butoxycarbonyl (Boc);1,1-carbonyldiimidazole (CDI); di-tert-butyl dicarbonate (BOC₂O);benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphoniumhexafluorophosphate (BOP); dichloromethane (DCM); diethyl ether (Et₂O);p-toluene sulfonic acid (PTSA); ethyl acetate (EtOAc); ethanol (EtOH);lithium bis(trimethylsilyl)amide (LHMDS); diisopropyl azodicarboxylate(DIAD); N,N-diisopropyl-ethylamine (DIEA or DIPEA);N,N-dimethylformamide (DMF); dimethyl sulfoxide (DMSO);diphenylphosphoryl azide (DPPA); hour(s) (h);2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU); High Performance Liquid Chromatography(HPLC); lithium aluminium hydride (LAH); liquid chromatography coupledwith mass spectrometry (LCMS); lithium diisopropylamide (LDA); methanol(MeOH); milliliter(s) (mL); minute(s) (min); microwave (MW);n-butyllithium (n-BuLi);1,1-bis(diphenylphosphino)-ferrocenedichloropalladium (II)(PdCl₂(dppf)); tris(dibenzylideneacetone)dipalladium (0) (Pd₂(dba)₃);dichlorobis(triphenylphosphine)palladium (II) (PdCl₂(PPh₃)₂); roomtemperature (RT); trifluoroacetic acid (TFA); tetrahydrofuran (THF);thin layer chromatography (TLC); retention time (t_(R)); &4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (Xantophos).

Intermediate 16-chloro-3-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrazin-2-amine

Step a: To a solution of 3-bromo-2-(trifluoromethyl)pyridine (1.0 g,4.42 mmol), XantPhos (256 mg, 0.442 mmol), Pd₂(dba)₃ (203 mg, 0.221mmol) in dioxane (12 mL) was added (at RT and under N₂)2-ethylhexyl-3-mercaptopropanoate (1.1 mL, 4.87 mmol) followed byaddition of DIPEA (1.55 mL, 8.85 mmol). The resulting solution wasstirred in a microwave reactor for 1 h at 110° C. After cooling to RT,the reaction was filtered through a pad of Celite followed by EtOAc (25mL) wash. The combined filtrates were concentrated and the resultingresidue was purified by silica chromatography (0 to 30% gradient ofEtOAc/heptane) to give 2-ethylhexyl3-((2-(trifluoromethyl)pyridin-3-yl)thio)propanoate (1.41 g, 3.88 mmol).MS m/z 364.0 (M+H)⁺.

Step b: To a solution of 2-ethylhexyl3-((2-(trifluoromethyl)pyridin-3-yl)thio)propanoate (1.0 g, 2.75 mmol)in THF (8 mL) was added at −78° C. and under N₂ potassium tert-butoxide(1 M in THF, 8.25 mL, 8.25 mmol). After stirring vigorously at −78° C.for 20 min, the reaction was quenched with K₂CO₃ aq (2 M, 500 μL) andthe volatiles were removed under reduced pressure. The resulting residuewas poured into a separation funnel containing K₂CO₃ aq (2 M, 30 mL) andit was extracted with Et₂O (2×20 mL). The aqueous phase was acidifiedwith 6 M HCl until pH 4 and the resulting cloudy suspension wasextracted with CHCl₃:IPA (9:1; 3×20 mL) to give2-(trifluoromethyl)pyridine-3-thiol (380 mg, 2.12 mmol). MS m/z 180.0(M+H)⁺.

Step c: To a solution of 2-(trifluoromethyl)pyridine-3-thiol (285 mg,1.591 mmol), 3-bromo-6-chloropyrazin-2-amine (414 mg, 1.988 mmol),XantPhos (101 mg, 0.175 mmol), and Pd₂(dba)₃ (72.8 mg, 0.08 mmol) indioxane (2 mL) was added (at RT and under N₂) DIPEA (556 μL, 3.18 mmol).The resulting solution was stirred in a microwave reactor for 1.5 h at130° C. After cooling to RT, the reaction was diluted with EtOAc and itwas filtered through a pad of Celite followed by EtOAc (25 mL) wash. Thecombined filtrates were concentrated and the resulting residue waspurified by silica chromatography (0 to 30% gradient of EtOAc/heptane)to give6-chloro-3-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrazin-2-amine (1.41g, 3.88 mmol). ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 8.64 (dd, J=4.55,1.26 Hz, 1H), 7.90 (s, 1H), 7.82 (dd, J=8.08, 0.76 Hz, 1H), 7.46 (dd,J=8.08, 4.80 Hz, 1H); ¹⁹F NMR (376 MHz, CHLOROFORM-d) δ ppm −64.34 (s,1F). MS m/z 307.1 (M+H)⁺.

Intermediate 26-(4-amino-4-methylpiperidin-1-yl)-3-(2,3-dichlorophenyl)pyrazin-2-amine

A suspension of 3-bromo-6-chloropyrazin-2-amine (1.2 g, 5.76 mmol),(2,3-dichlophenyl)boronic acid (1.1 g, 5.76 mmol), potassium phosphate(3.67 g, 17.27 mmol), and PdCl₂(dppf) DCM adduct (235 mg, 0.288 mmol) inMeCN:H₂O (9:1, 15 mL, degassed) was stirred in a microwave reactor for 4h at 120° C. After cooling to RT, the reaction was filtered through apad of Celite followed by EtOAc (25 mL) wash. The combined filtrateswere concentrated and the resulting residue was purified by silicachromatography (0 to 30% gradient of EtOAc/heptane) to give6-(4-amino-4-methylpiperidin-1-yl)-3-(2,3-dichlorophenyl)pyrazin-2-amine(633 mg, 2.306 mmol). MS m/z 276.4 (M+H)⁺.

Intermediate 3 6-chloro-3-((2,3-dichlorophenyl)thio)pyrazin-2-amine

A mixture of 3-bromo-6-chloropyrazin-2-amine (5.0 g, 23.99 mmol),2,3-dichlorobenzenethiol (6.44 g, 36.0 mmol), copper(I)iodide (914 mg,4.80 mmol), potassium phosphate (10.18 g, 48.0 mmol), and1,10-phenanthroline (1.73 mg, 9.59 mmol) in dioxane (50 mL, degassed)was stirred for 16 h at 85° C. After cooling to RT, the reaction wasdiluted with EtOAc, and it was filtered through a pad of Celite followedby EtOAc (50 mL) wash. The volatiles were removed under reduced pressureand the resulting residue was purified by silica chromatography (0 to25% gradient of DCM/toluene) to give6-chloro-3-((2,3-dichlorophenyl)thio)pyrazin-2-amine (3.7 g, 12.07mmol). MS m/z 306.0 (M+H)⁺.

Intermediate 4 (R) and(S)-2-(7-azaspiro[3.5]nonan-1-yl)isoindoline-1,3-dione

Step a: A suspension of tert-butyl1-amino-7-azaspiro[3.5]nonane-7-carboxylate (250 mg, 1.04 mmol),phthalic anhydride (193 mg, 1.3 mmol), activated molecular sieves (3angstroms, 250 mg), and DIPEA (545 μL, 3.12 mmol) in toluene (4 mL) wasstirred for 16 h at 105° C. After cooling to RT, the mixture wasfiltered through a pad of Celite followed by EtOAc (10 mL) wash. Thevolatiles were removed under reduced pressure and the resulting residuewas purified by silica chromatography (5 to 40% gradient ofEtOAc/heptane) to give tert-butyl1-(1,3-dioxoisoindolin-2-yl)-7-azaspiro[3.5]nonane-7-carboxylate (233mg, 0.629 mmol). MS m/z 370.4 (M+H)⁺.

Step b: A solution of tert-butyl1-(1,3-dioxoisoindolin-2-yl)-7-azaspiro[3.5]nonane-7-carboxylate (233mg, 0.629 mmol) and HCl (4 M in dioxane, 800 μL, 3.21 mmol) in dioxane(5 mL) was stirred for 16 h at RT. The volatiles were removed on therotavap to give the HCl salt of the title compound (195 mg, 0.636 mmol).MS m/z 270.3 (M+H)⁺.

Step c: Chiral SFC purification was performed under the followingconditions; column: Cellulose LUX-2 21×250 mm, flow rate: 75 g perminute, mobile phase: 50% MeOH and 10 mM NH₄OH in CO₂, detection: 220 nmUV to obtain two peaks R_(t)(P1)=3.6 min (enantiomer R); R_(t) (P2)=7.4min (enantiomer S).

Intermediate 52-(1,1-dioxido-1-thia-8-azaspiro[4.5]decan-4-yl)isoindoline-1,3-dione

Step a: A solution of8-(tert-butoxycarbonyl)-1-thia-8-azaspiro[4.5]decane-4-carboxylic acid1,1-dioxide (prepared from tert-butyl 4-oxopiperidine-1-carboxylate in 4steps as described in Carreira et al., Org Lett., 2011, 13, 6134-6136;2.00 g, 6.00 mmol), diphenyl phosphorazidate (2.0 g, 7.26 mmol), andEt₃N (1.0 mL, 7.26 mmol) in toluene (37 mL) was stirred for 1.5 h at115° C. Benzyl alcohol (1.50 mL, 14.52 mmol) was added and the resultingmixture was stirred for 16 h at 100° C. After cooling to RT, thereaction mixture was poured into a separation funnel containing NaHCO₃sat. aq. (30 mL) and it was extracted with EtOAc (3×20 mL). The combinedorganic phases were dried over MgSO₄, filtered and the volatiles wereremoved under reduced pressure. The resulting residue was purified bysilica chromatography (10 to 90% gradient of EtOAc/heptane) to givetert-butyl4-(((benzyloxy)carbonyl)amino)-1-thia-8-azaspiro[4.5]decane-8-carboxylate1,1-dioxide (1.57 g. 3.58 mmol) as a white solid. MS m/z 339.4 (M+H)⁺.

Step b: A suspension of tert-butyl4-(((benzyloxy)carbonyl)amino)-1-thia-8-azaspiro[4.5]decane-8-carboxylate1,1-dioxide (570 mg. 1.30 mmol) and Pd/C (10% on charcoal, 138 mg) inTHF (8 mL) was vigorously stirred under H₂ atmosphere for 16 h. Thereaction was filtered through a plug of Celite followed by EtOAc (20 mL)wash. The volatiles were removed under reduced pressure to givetert-butyl 4-amino-1-thia-8-azaspiro[4.5]decane-8-carboxylate1,1-dioxide which was used in next step without further purification.

Step c: A suspension of tert-butyl4-amino-i-thia-8-azaspiro[4.5]decane-8-carboxylate 1,1-dioxide (415 mg,1.363 mmol), phthalic anhydride (252 mg, 1.704 mmol), and activatedmolecular sieves (3 angstroms, 500 mg) in toluene (7 mL) was vigorouslystirred for 16 h at 115° C. After cooling to RT, the mixture wasfiltered through a pad of Celite followed by EtOAc (10 mL) wash, and thevolatiles were removed under reduced pressure. The resulting residue waspurified by silica chromatography (0 to 10% gradient of MeOH/DCM) togive tert-butyl4-(1,3-dioxoisoindolin-2-yl)-1-thia-8-azaspiro[4.5]decane-8-carboxylate1,1-dioxide (385 mg, 0.886 mmol) as a white foam. MS m/z 433.1 (M−H)⁻.

Step d: A solution of tert-butyl4-(1,3-dioxoisoindolin-2-yl)-1-thia-8-azaspiro[4.5]decane-8-carboxylate1,1-dioxide (385 mg, 0.886 mmol) and HCl (4 M in dioxane, 2.22 mL, 8.86mmol) in dioxane (4 mL) was stirred for 16 h at RT. The mixture wasdiluted with dioxane (20 mL) and filtered to give2-(1,1-dioxido-1-thia-8-azaspiro[4.5]decan-4-yl)isoindoline-1,3-dione(HCl salt, 328 mg, 0.884 mmol) as a white solid. MS m/z 335.4 (M+H)⁺.

Intermediate 6(R)-2-methyl-N—((S)-7-azaspiro[3.5]nonan-1-yl)propane-2-sulfinamide

Step a: A solution of tert-butyl1-oxo-7-azaspiro[3.5]nonane-7-carboxylate (5.24 g, 21.9 mmol),titanium(IV) isopropoxide (16.2 mL, 54.7 mmol), and(R)-2-methylpropane-2-sulfinamide (3.45 g, 28.5 mmol) in THF (99 mL) wasstirred for 12 h at 65° C. After cooling to −78° C., MeOH (9.9 mL) wasadded followed by lithium borohydride (1.43 g, 65.7 mmol). The resultingmixture was stirred at −78° C. for 3 h and at RT for 1 h. MeOH wasslowly added to quench the excess of borohydride followed by addition ofbrine. The resulting mixture was stirred for 15 min and then filteredthrough Celite. The aqueous mixture was extracted with EtOAc (3×20 mL).The organic phases were dried over MgSO₄, filtered, and the volatileswere removed under reduced pressure. The resulting residue was purifiedby silica chromatography (0 to 50% gradient of EtOAc/heptane to give(S)-tert-butyl1-((R)-1,1-dimethylethylsulfinamido)-7-azaspiro[3.5]nonane-7-carboxylate(4.79 g. 13.90 mmol) as a white solid. MS m/z 345.3 (M+H)⁺.

Step b: A solution of (S)-tert-butyl1-((R)-1,1-dimethylethylsulfinamido)-7-azaspiro[3.5]nonane-7-carboxylate(0.4 g, 1.16 mmol) and TFA (450 μL, 5.81 mmol) in DCM (3.5 mL) wasstirred for 30 min at 40° C. Na₂CO₃ sat. aq. was added until pH 11 andthe aqueous mixture was extracted with DCM (3×15 mL). The combinedorganic phases were washed with brine, dried over Na₂SO₄, filtered, andthe volatiles were removed under reduced pressure to give(R)-2-methyl-N—((S)-7-azaspiro[3.5]nonan-1-yl)propane-2-sulfinamide (237mg, 0.97 mmol) as a white solid. MS m/z 245.5 (M+H)⁺.

Intermediate 7 N-(4-methoxybenzyl)-8-azaspiro[4.5]decan-1-amine

Step a: To a solution of tert-butyl1-oxo-8-azaspiro[4.5]decane-8-carboxylate (1.8 g, 7.11 mmol), and(4-methoxyphenyl)methanamine (1.07 g, 7.82 mmol) in DCE (7 mL) was addedsodium cyanoborohydride (2.23 g, 35.5 mmol) in portions and stirred atRT for 65 h. The mixture was diluted with saturated aqueous sodiumbicarbonate solution (10 mL) and extracted with EtOAc (3×20 mL) Thecombined organic phases were washed with brine and concentrated. Theresulting residue was purified by silica chromatography (0 to 2%gradient of MeOH/DCM, 0.25% Et₃N modified, followed by 0 to 50% gradientof EtOAc/heptane) to give tert-butyl1-((4-methoxybenzyl)amino)-8-azaspiro[4.5]decane-8-carboxylate (1.1 g,2.94 mmol) as a colorless wax. MS m/z 375.3 (M+H)⁺.

Step b: A solution of tert-butyl1-((4-methoxybenzyl)amino)-8-azaspiro[4.5]decane-8-carboxylate (1.1 g,2.94 mmol) and TFA (2 mL) in DCM (2 mL) was stirred for 15 min at 0° C.The volatiles were removed under reduced pressure. The resulting residuewas diluted with aqueous NaHCO₃ (10 mL) and extracted with EtOAc (4×10mL) to give N-(4-methoxybenzyl)-8-azaspiro[4.5]decan-1-amine (0.8 g,2.92 mmol) as a colorless oil. MS m/z 275.2 (M+H)⁺.

N-(4-methoxybenzyl)-3-azaspiro[5.5]undecan-7-amine was obtainedfollowing the above procedure starting from tert-butyl7-oxo-3-azaspiro[5.5]undecane-3-carboxylate.

Intermediate 8N—((R)-1-(4-methoxyphenyl)ethyl)-8-azaspiro[4.5]decan-1-amine

Step a: To a solution of tert-butyl1-oxo-8-azaspiro[4.5]decane-8-carboxylate (1.15 g, 4.54 mmol), and(R)-1-(4-methoxyphenyl)ethanamine (961 mg, 6.36 mmol) in DCE (3 mL) wasadded sodium cyanoborohydride in portions and stirred for 16 h at RT.The mixture was diluted with saturated aqueous sodium bicarbonatesolution (5 mL) and extracted with EtOAc (3×10 mL). The combined organicphases were washed with brine and concentrated. The resulting residuecontaining a 9:1 mixture of diastereomers was purified by silicachromatography (0 to 20% gradient of EtOAc/heptane) to give tert-butyl1-(((R)-1-(4-methoxyphenyl)ethyl)amino)-8-azaspiro[4.5]decane-8-carboxylate(major diastereomer; 431 mg, 1.11 mmol) pure. ¹H NMR (400 MHz, DMSO-d₆)δ ppm 7.18-7.24 (m, 2H), 6.81-6.86 (m, 2H), 3.76 (d, J=13.64 Hz, 1H),3.72 (s, 3H), 3.64-3.70 (m, 2H), 2.65-2.92 (m, 2H), 2.05-2.14 (m, 1H),1.80-1.91 (m, 1H), 1.65-1.75 (m, 1H), 1.42-1.60 (m, 4H), 1.40 (s, 9H),1.28-1.35 (m, 1H), 1.20 (d, J=6.57 Hz, 3H), 1.09-1.17 (m, 2H), 0.80 (d,J=11.37 Hz, 1H). MS m/z 389.6 (M+H)⁺.

Step b: To a solution of tert-butyl1-(((R)-1-(4-methoxyphenyl)ethyl)amino)-8-azaspiro[4.5]decane-8-carboxylate(major diastereomer; 431 mg, 1.11 mmol) in DCM (2 mL) was added TFA (2mL) and stirred for 10 min at RT. The reaction was concentrated withfurther addition of DCM, then diluted with saturated aqueous NaHCO₃ andextracted with DCM (3×20 mL). Washed organics with brine, dried overNa₂SO₄, filtered and concentrated to giveN—((R)-1-(4-methoxyphenyl)ethyl)-8-azaspiro[4.5]decan-1-amine. ¹H NMR(400 MHz, METHANOL-d₄) δ 7.25 (d, J=8.6 Hz, 2H), 6.87 (d, J=8.7 Hz, 2H),3.85-3.78 (m, 1H), 3.78 (s, 3H), 3.35 (m, 1H), 3.28 (m, 1H), 3.03 (m,2H), 2.63 (dd, J=9.6, 7.3 Hz, 1H), 2.06-1.85 (m, 2H), 1.83-1.69 (m, 2H),1.62 (m, 1H), 1.54-1.38 (m, 4H), 1.33 (d, J=6.6 Hz, 3H), 1.31-1.23 (m,1H). MS m/z 289.5 (M+H)⁺.

Intermediate 9N—((R)-1-(4-methoxyphenyl)ethyl)-1-oxa-8-azaspiro[4.5]decan-4-amine

Step a: To a solution of tert-butyl4-oxo-1-oxa-8-azaspiro[4.5]decane-8-carboxylate (prepared fromtert-butyl 4-oxopiperidine-1-carboxylate in 3 steps as described inCarreira et al., Org Lett., 2013, 15, 4766-4769; 200 mg, 0.78 mmol), and(R)-1-(4-methoxyphenyl)ethanamine (474 mg, 3.13 mmol) in DCE (1 mL) wasadded sodium cyanoborohydride in portions (393 mg, 3.13 mmol). Theresulting reaction was stirred for 16 h at RT. Lithium borohydride (34mg, 1.6 mmol) was added and the mixture was stirred for 30 min at RT.The mixture was diluted with MeOH (2 mL) and the volatiles were removedunder reduced pressure (two times). NaHCO₃ sat. aq. (5 mL) was added andthe mixture was extracted with DCM (3×20 mL). The combined organicphases were dried over Na₂SO₄, filtered, and the volatiles were removedunder reduced pressure. The resulting 9:1 mixture of diastereomers waspurified by silica chromatography (0 to 40% gradient of EtOAc/heptane)to give tert-butyl4-(((R)-1-(4-methoxyphenyl)ethyl)amino)-1-oxa-8-azaspiro[4.5]decane-8-carboxylate(65 mg, 0.17 mmol) diastereomerically pure. Major diastereomer: ¹H NMR(400 MHz, CDCl₃) δ ppm 7.15 (d, J=8.6 Hz, 2H), 6.79 (d, J=8.6 Hz, 2H),3.96-3.76 (m, 2H), 3.77-3.66 (m, 5H), 3.60 (t, J=8.1 Hz, 1H), 2.98 (m,2H), 2.76 (t, J=7.8 Hz, 1H), 1.95 (m, 1H), 1.67-1.41 (m, 4H), 1.40 (s,9H), 1.33 (d, J=3.1 Hz, 1H), 1.21 (d, J=6.5 Hz, 3H), 1.08-0.92 (m, 1H).MS m/z 391.6 (M+H)⁺.

Step b: A solution of tert-butyl4-(((R)-1-(4-methoxyphenyl)ethyl)amino)-1-oxa-8-azaspiro[4.5]decane-8-carboxylate(major diastereomer, 65 mg, 0.17 mmol) and TFA (2 mL) in DCM (2 mL)stirred for 10 min at RT. The volatiles were removed under reducedpressure, diluted with NaHCO₃ sat. aq. (5 mL), and extracted with DCM(3×20 mL). The combined organic phases were washed with brine, driedover Na₂SO₄, filtered and the volatiles were removed under reducedpressure to giveN—((R)-1-(4-methoxyphenyl)ethyl)-1-oxa-8-azaspiro[4.5]decan-4-amine (40mg, 0.13 mmol) which was used without further purification. MS m/z 291.5(M+H)⁺.

Intermediate 103-((2-amino-3-chloropyridin-4-yl)thio)-6-chloropyrazin-2-amine

Step a: Commercially available 2,3-dichloro-4-iodopyridine was convertedto 3-chloro-4-iodopyridin-2-amine by procedure a, as described in Marieet al., Molecules, 2012, 17, 10683-10707.

Step b: To a solution of 3-amino-5-chloropyrazine-2-thiol (100 mg, 0.619mmol), 3-chloro-4-iodopyridin-2-amine (315 mg, 1.238 mmol), XantPhos(35.8 mg, 0.062 mmol), and Pd₂(dba)₃ (28.3 mg, 0.03 mmol) in dioxane (3mL) was added (at RT and under N₂) DIPEA (324 μL, 1.856 mmol). Theresulting solution was stirred in a microwave reactor for 2.5 h at 100°C. After cooling to RT, the reaction was diluted with EtOAc and it wasfiltered through a pad of Celite followed by EtOAc (10 mL) wash. Thecombined filtrates were concentrated and the resulting residue waspurified by silica chromatography (0 to 5% gradient of MeOH/DCM) to give3-((2-amino-3-chloropyridin-4-yl)thio)-6-chloropyrazin-2-amine (1.41 g,3.88 mmol). ¹H NMR (400 MHz, METHANOL-d₄) δ ppm 7.88 (s, 1H), 7.68 (d,J=5.56 Hz, 1H), 6.06 (d, J=5.56 Hz, 1H), 1.35-1.43 (m, 2H). MS m/z 288.2(M+H)⁺.

Intermediate 11 3-amino-5-chloropyrazine-2-thiol

Step a: A solution of 3-bromo-6-chloropyrazin-2-amine (4.95 g, 23.74mmol) in dioxane (119 mL) was sparged with nitrogen for 10 min. Then,2-ethylhexyl 3-mercaptopropanoate (3.79 mL, 24.92 mmol), Xantphos (1.37g, 2.37 mmol), Pd₂(dba)₃ (1.08 g, 1.19 mmol), and DIPEA (8.29 mL, 47.5mmol) were added. The resulting mixture was stirred at 105° C. for 24 hand the reaction mixture was filtered through Celite and concentrated.The crude was purified by silica chromatography (0-40% gradient ofEtOAc/heptane) to afford 2-ethylhexyl3-((3-amino-5-chloropyrazin-2-yl)thio)propanoate (6.24 g, 18.04 mmol) asa yellow oil. ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 7.82 (s, 1H), 4.93(br. s., 2H), 4.14-3.96 (m, 2H), 3.47 (t, J=6.9 Hz, 2H), 2.78 (t, J=6.9Hz, 2H), 1.67-1.51 (m, 1H), 1.44-1.20 (m, 8H), 0.90 (t, J=7.4 Hz, 6H).MS m/z 346.0 (M+H)⁺.

Step b: To a solution of 2-ethylhexyl3-((3-amino-5-chloropyrazin-2-yl)thio)propanoate (2.3 g, 6.65 mmol) inTHF (33 mL) at −78° C., potassium tert-butoxide (1 M in THF, 19.95 mL,19.95 mmol) was added and the resulting mixture was stirred at −78° C.for 1 h. MeOH (20 mL) was added and the resulting mixture wasconcentrated. The crude was dissolved in MeOH, filtered, and waspurified by HPLC (gradient elution 5-20%, acetonitrile in water, 0.1%TFA modifier) to afford 3-amino-5-chloropyrazine-2-thiol (TFA salt: 1.3g, 4.72 mmol) as a yellow solid. MS m/z 162.0 (M+H)⁺.

Intermediate 12 6-chloro-3-((3-chloropyridin-4-yl)thio)pyrazin-2-amine

A solution of 3-amino-5-chloropyrazine-2-thiol (TFA salt: 0.158 g, 0.978mmol) in dioxane (4.9 mL) was sparged with nitrogen for 10 min. Then,3-chloro-4-iodopyridine (0.468 g, 1.955 mmol), Xantphos (0.057 g, 0.098mmol), Pd₂(dba)₃ (0.045 g, 0.049 mmol), and DIPEA (0.512 mL, 2.93 mmol)were added. The resulting mixture was stirred at 105° C. for 10 h,filtered through Celite and concentrated. The crude was purified bysilica chromatography (0-40% gradient of EtOAc/heptane; heptanecontaining 2% of Et₃N) to afford6-chloro-3-((3-chloropyridin-4-yl)thio)pyrazin-2-amine (75 mg, 0.274mmol) as a white solid. ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 8.46 (s,1H), 8.22 (d, J=5.3 Hz, 1H), 7.96 (s, 1H), 6.68 (d, J=5.3 Hz, 1H), 5.17(br. s., 2H). MS m/z 273.0 (M+H)⁺.

Intermediate 13 6-chloro-3-((2-chloropyridin-3-yl)thio)pyrazin-2-amine

A solution of 3-amino-5-chloropyrazine-2-thiol (TFA salt: 0.2 g, 1.238mmol) in dioxane (6.2 mL) was sparged with nitrogen for 10 min. Then,2-chloro-3-iodopyridine (0.593 g, 2.475 mmol), Xantphos (0.072 g, 0.124mmol), Pd₂(dba)₃ (0.057 g, 0.062 mmol), and DIPEA (0.65 mL, 3.71 mmol)were added. The resulting mixture was stirred at 105° C. for 10 h,filtered through Celite and concentrated. The crude was purified bysilica chromtography (0-40% gradient of EtOAc/heptane, containing 2% ofEt₃N) to afford 6-chloro-3-((2-chloropyridin-3-yl)thio)pyrazin-2-amine(95 mg, 0.348 mmol) as a white solid. ¹H NMR (400 MHz, CHLOROFORM-d) δppm 8.28-8.38 (m, 1H), 7.91 (s, 1H), 7.51-7.59 (m, 1H), 7.22 (dd, J=7.9,4.6 Hz, 1H), 5.25 (br. s., 2H). MS m/z 273.0 (M+H)⁺.

Intermediate 146-chloro-3-((2,3-dichloropyridin-4-yl)thio)pyrazin-2-amine

A solution of 3-amino-5-chloropyrazine-2-thiol (TFA salt: 0.50 g, 1.814mmol) in dioxane (90 mL) was degassed with nitrogen for 10 min. Then,2,3-dichloro-4-iodopyridine (0.0.99 g, 3.63 mmol), Xantphos (0.105 g,0.181 mmol), Pd₂(dba)₃ (0.083 g, 0.091 mmol), and DIPEA (0.95 mL, 5.44mmol) were added. The resulting mixture was stirred at 105° C. for 10 h,filtered through Celite and concentrated. The crude was purified bysilica chromatography (0-10% gradient of EtOAc/DCM). ¹H NMR (400 MHz,DMSO-d₆) δ ppm 8.13 (d, J=5.3 Hz, 1H), 7.95 (s, 1H), 7.30 (br. s, 2H),6.83 (d, J=5.3 Hz, 1H). MS m/z 306.9 (M+H)⁺.

Intermediate 156-chloro-3-((3-chloro-2-fluoropyridin-4-yl)thio)pyrazin-2-amine

A solution of 3-amino-5-chloropyrazine-2-thiol (TFA salt: 50 mg, 0.181mmol) in dioxane (1.8 mL) was sparged with nitrogen for 10 min. Then,3-chloro-2-fluoro-4-iodopyridine (0.140 g, 0.544 mmol), Xantphos (11 mg,0.018 mmol), Pd₂(dba)₃ (8 mg, 0.009 mmol), and DIPEA (95 μL, 0.544 mmol)were added. The resulting mixture was stirred at 100° C. for 10 h,filtered through Celite, and concentrated. The crude was purified bysilica chromatography (0 to 40% gradient of EtOAc/heptane; heptanecontaining 2% Et₃N) to afford6-chloro-3-((3-chloro-2-fluoropyridin-4-yl)thio)pyrazin-2-amine (41 mg,0.137 mmol) as a white solid. ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 8.06(s, 1H), 7.91 (d, J=5.3 Hz, 1H), 6.63 (d, J=5.3 Hz, 1H), 5.30 (br. s,2H). MS m/z 291.0 (M+H)⁺.

Intermediate 17 2-(2-oxa-8-azaspiro[4.5]decan-4-yl)isoindoline-1,3-dione

Following procedures of Dirat et al., PCT Int. Appl., 20044078750, 16Sep. 2004, prepared tert-butyl4-hydroxy-2-oxa-8-azaspiro[4.5]decane-8-carboxylate. ¹H NMR (400 MHz,Chloroform-d) δ 4.13 (dd, J=10.1, 4.6 Hz, 1H), 4.03 (dd, J=4.6, 2.0 Hz,1H), 3.78-3.71 (m, 2H), 3.69 (d, J=8.6 Hz, 1H), 3.67-3.58 (m, 2H), 3.29(m, 1H), 3.16 (m, 1H), 1.78 (m, 2H), 1.58 (m, 1H), 1.50 (m, 2H), 1.47(s, 9H). MS m/z 258.1 (M−H)⁺ from 1-tert-butyl 4-ethylpiperidine-1,4-dicarboxylate in four steps, then converted to2-(2-oxa-8-azaspiro[4.5]decan-4-yl)isoindoline-1,3-dione in two steps asfollows.

Step a: To a solution of tert-butyl4-hydroxy-2-oxa-8-azaspiro[4.5]decane-8-carboxylate (306 mg, 1.19 mmol),phthalimide (262 mg, 1.78 mmol) and triphenylphosphine (468 mg, 1.78mmol) in THF (10 mL) was added diisopropylazadicarboxylate (0.374 mL,1.78 mmol) and stirred 16 h. Concentrated and purified by silicachromatography (0 to 50% gradient of ethyl acetate/heptane) to obtainracemic tert-butyl4-(1,3-dioxoisoindolin-2-yl)-2-oxa-8-azaspiro[4.5]decane-8-carboxylate(190 mg, 0.49 mmol). ¹H NMR (400 MHz, Chloroform-d) δ 7.88 (dd, J=5.4,3.0 Hz, 2H), 7.77 (dd, J=5.5, 3.0 Hz, 2H), 4.65 (dd, J=8.7, 5.6 Hz, 1H),4.40 (dd, J=9.5, 5.6 Hz, 1H), 4.26 (t, J=9.0 Hz, 1H), 4.08 (d, J=8.5 Hz,1H), 3.98 (d, J=8.5 Hz, 1H), 3.84 (m, 1H), 3.58 (m, 1H), 3.20 (m, 1H),2.94 (m, 1H), 1.73 (m, 2H), 1.56 (s, 9H), 1.42-1.36 (m, 2H).

Step b: To a solution of racemic tert-butyl4-(1,3-dioxoisoindolin-2-yl)-2-oxa-8-azaspiro[4.5]decane-8-carboxylate(190 mg, 0.49 mmol) in dichloromethane (3 mL) was added trifluoroaceticacid (1 mL). Concentrated with further addition of dichloromethane, thenacetonitrile to obtain2-(2-oxa-8-azaspiro[4.5]decan-4-yl)isoindoline-1,3-dione as a TFA salt(quantitative). MS m/z 287.0 (M+H)⁺. Used without furthercharacterization.

Intermediate 18 2-chloro-3-mercaptobenzamide

Step a: A mixture of 2-chloro-3-fluorobenzonitrile (3.15 g, 20.25 mmol),2-methylpropane-2-thiol (2.283 mL, 20.25 mmol) and Cs₂CO₃ (6.598 g,20.25 mmol) in DMF (100 mL) was stirred for 48 h at 22° C. The reactionmixture was diluted with water (200 mL) and EtOAc (300 mL). EtOAc layerwas washed with water (3×300 mL), brine (3×100 mL), dried over Na₂SO₄,filtered and concentrated. The resulting residue was purified by HPLC(gradient elution: 45 to 70% acetonitrile in water, 5 mM NH₄OHmodifier), to give 3-(tert-butylthio)-2-chlorobenzonitrile (1.33 g, 5.89mmol). MS m/z 226.1 (M+H)⁺.

Step b: A mixture of 3-(tert-butylthio)-2-chlorobenzonitrile (217 mg,0.961 mmol) and NaOH (1 N, 2.88 mL, 2.88 mmol) in MeOH (11 mL) wasirradiated in a microwave reactor for 35 min at 90° C. After cooling toRT, the reaction was concentrated and dissolved in MeOH. The solid wasfiltered off and the filtrate was concentrated to almost dryness and waspurified by HPLC (gradient elution: 25 to 50% acetonitrile in water, 5mM NH₄OH modifier), to give 3-(tert-butylthio)-2-chlorobenzamide (93.6mg, 0.384 mmol). MS m/z 244 (M+H)⁺.

Step c: A mixture of 3-(tert-butylthio)-2-chlorobenzamide (190 mg, 0.779mmol) and conc. HCl (2.36 mL, 78 mmol) was stirred for 45 min at 85° C.After cooling to RT, the reaction was concentrated to dryness to yieldcrude 2-chloro-3-mercaptobenzamide (HCl salt: 156 mg, 0.651 mmol). MSm/z 188 (M+H)⁺.

Intermediate 192-(8-(6-amino-5-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrazin-2-yl)-2-oxa-8-azaspiro[4.5]decan-4-yl)isoindoline-1,3-dione

Coupled 2-(2-oxa-8-azaspiro[4.5]decan-4-yl)isoindoline-1,3-dione TFAsalt with6-chloro-3-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrazin-2-amine (151mg, 0.492 mmol) using the standard procedures described above. Dilutedwith DCM and purified by silica chromatography (0 to 60% gradient ofethyl acetate/heptane) to obtain2-(8-(6-amino-5-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrazin-2-yl)-2-oxa-8-azaspiro[4.5]decan-4-yl)isoindoline-1,3-dione(0.140 g, 0.178 mmol). MS m/z 557.1 (M+H)⁺. Chiral SFC purificationperformed as follows; column: OJ-H 21×250 mm, flow rate: 80 g perminute, mobile phase: 45% MeOH and 5 mM NH₄OH in CO₂, detection: masstriggered to obtain single enantiomers peak 1 (P1), R_(t): 2.77 min. MSm/z 557.1 (M+H)*, and peak 2 (P2), R_(t): 3.91 min. MS m/z 557.2 (M+H)⁺.Phthalimide deprotection performed on each enantiomer separately withoutfurther characterization.

Intermediate 20 3-chloro-4-iodo-N,N-dimethylpyridin-2-amine

A solution of 3-chloro-2-fluoro-4-iodopyridine (0.26 g, 1.01 mmol) anddimethylamine (2 M in THF, 1.5 ml, 3.03 mmol) in DMSO (3.4 mL) wasstirred at for 2 h at 70° C. After cooling to RT, water was added andthe aqueous mixture was extracted with EtOAc. The combined organicphases were washed with water, brine, dried with Na₂SO₄, filtered andconcentrated under reduced pressure to give3-chloro-4-iodo-N,N-dimethylpyridin-2-amine (0.26 g, 0.922 mmol) as acolorless oil. ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 7.75 (d, J=5.3 Hz,1H), 7.33 (d, J=5.0 Hz, 1H), 3.00 (s, 6H). MS m/z 282.9 (M+H)⁺.

Intermediate 21 3-chloro-4-iodo-2-methoxypyridine

A solution of 3-chloro-2-fluoro-4-iodopyridine (150 mg, 0.571 mmol) andNaOMe (0.5 M in MeOH, 3.4 ml, 1.71 mmol) in DMSO (1.9 mL) was stirred atfor 1 h at 70° C. After cooling to RT, water was added and the aqueousmixture was extracted with EtOAc. The combined organic phases werewashed with water, brine, dried over Na₂SO₄, filtered, and concentratedunder reduced pressure to give 3-chloro-4-iodo-2-methoxypyridine (123mg, 0.456 mmol) as a colorless oil. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 7.81(d, J=5.3 Hz, 1H), 7.55 (d, J=5.3 Hz, 1H), 3.92 (s, 3H). MS m/z 269.9(M+H)⁺.

Intermediate 226-chloro-3-((3-(trifluoromethyl)pyridin-4-yl)thio)pyrazin-2-amine

A mixture of 3-amino-5-chloropyrazine-2-thiol (750 mg, 4.09 mmol),4-bromo-3-(trifluoromethyl)pyridine (1.63 g, 5.31 mmol), Xantphos (236mg, 0.409 mmol), Pd₂(dba)₃ (187 mg, 0.204 mmol), and DIPEA (2.14 mL,12.26 mmol) in dioxane (degassed, 50 mL) was stirred for 16 h at 100° C.After cooling to RT, the reaction was filtered through a pad of Celitefollowed by EtOAc (25 mL) wash. The combined filtrates were concentratedunder reduced pressure and the resulting residue was purified by silicachromatography (0 to 40% gradient of EtOAc/DCM) to give6-chloro-3-((3-(trifluoromethyl)pyridin-4-yl)thio)pyrazin-2-amine (722mg, 2.35 mmol) as a light yellow solid. MS m/z 307.0 (M+H)⁺.

The following compounds were synthesized using the above procedure ormodifications to the above procedure using the corresponding iodo- orbromo-pyridyl and thiolate.

TABLE 1

Intermediate 23 6-chloro-3-((3-chloropyridazin-4-yl)thio)pyrazin-2-amine

A mixture of 3-amino-5-chloropyrazine-2-thiol (100 mg, 0.545 mmol),3,4-dicholoropyridazine (81 mg, 0.545 mmol), and DIPEA (0.142 mL, 0.817mmol) in MeCN (5.5 mL) was stirred for 12 h at 50° C. After cooling toRT, the precipitate was collected by vacuum filtration to give6-chloro-3-((3-chloropyridazin-4-yl)thio)pyrazin-2-amine (101 mg, 0.368mmol) as a brown solid. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 8.90 (d, J=5.4Hz, 1H), 7.95 (s, 1H), 7.31 (s, 2H), 7.15 (d, J=5.3 Hz, 1H). MS m/z274.1 (M+H)⁺.

Intermediate 24 tert-butyl 1-oxo-8-azaspiro[4.5]dec-2-ene-8-carboxylate

Step a: A mixture of tert-butyl 4-formylpiperidine-1-carboxylate (35.0g, 164 mmol), lithium tert-butoxyde (15.77 g, 197 mmol), andallylbromide (11.54 mL, 189 mmol) in DMF (328 mL) was stirred for 1 h at0° C. The mixture was poured into a separation funnel containing sat. aqNH₄C1:H₂O (1:1, 500 mL) and it was extracted with Et₂O (5×50 mL). Thecombined organic phases were dried over MgSO₄, filtered, and thevolatiles were removed under reduced pressure. The resulting residue waspurified by silica chromatography (0 to 25% gradient of EtOAc/heptane)to give tert-butyl 4-allyl-4-formylpiperidine-1-carboxylate (24 g, 95mmol) as colorless oil. ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 9.52 (s,1H), 5.53-5.76 (m, 1H), 4.96-5.19 (m, 2H), 3.80 (br. s., 2H), 2.97 (t,J=11.49 Hz, 2H), 2.26 (d, J=7.33 Hz, 2H), 1.95 (dt, J=13.71, 3.13 Hz,2H), 1.38-1.58 (m, 11H).

Step b: To a solution of tert-butyl4-allyl-4-formylpiperidine-1-carboxylate (24 g, 95 mmol) in THF (300 mL)was added (at −78° C. and under N₂) vinyl magnesium bromide (1 M in THF,118 mL, 118 mmol). The resulting solution was slowly warmed up to RTwithin 1 h. The mixture was poured into a separation funnel containingsat. aq NH₄Cl (250 mL) and it was extracted with EtOAc (4×50 mL). Thecombined organic phases were dried over MgSO₄, filtered and thevolatiles were removed under reduced pressure to give tert-butyl4-allyl-4-(1-hydroxyallyl)piperidine-1-carboxylate (26.7 g, 95 mmol) ascolorless oil. ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 9.52 (s, 1H),5.56-5.75 (m, 1H), 5.05-5.18 (m, 2H), 3.80 (br. s., 2H), 2.97 (t,J=11.49 Hz, 2H), 2.26 (d, J=7.33 Hz, 2H), 1.96 (dt, J=13.83, 3.06 Hz,2H), 1.49-1.60 (m, 2H), 1.41-1.49 (m, 9H). This compound was used innext step without further purification.

Step c: A mixture of tert-butyl4-allyl-4-(1-hydroxyallyl)piperidine-1-carboxylate (26.7 g, 95 mmol) andDess-Martin periodinane (44.3 g, 105 mmol) in DCM (380 mL) was stirredfor 1 h at RT. The mixture was poured into a separation funnelcontaining sat. aq NaHCO₃:Na₂SO₃ (1:1, 300 mL) and it was extracted withDCM (4×50 mL). The combined organic phases were dried over MgSO₄,filtered, and the volatiles were removed under reduced pressure toprovide a white solid. This solid was suspended in heptane (250 mL) andsonicated for 5 min. The white suspension was filtered through a pad ofCelite and the volatiles were removed under reduced pressure to givetert-butyl 4-acryloyl-4-allylpiperidine-1-carboxylate (26.5 g, 95 mmol)as yellow oil. ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 6.81 (dd, J=16.93,10.36 Hz, 1H), 6.40 (dd, J=16.80, 1.89 Hz, 1H), 5.71 (dd, J=10.36, 2.02Hz, 1H), 5.46-5.66 (m, 1H), 4.91-5.14 (m, 2H), 3.78 (br. s., 2H), 2.96(br. s., 2H), 2.25-2.39 (m, 2H), 1.97-2.15 (m, 2H), 1.37-1.57 (m, 11H).This compound was used in next step without further purification.

Step d: To a solution of tert-butyl4-acryloyl-4-allylpiperidine-1-carboxylate (26.5 g, 95 mmol) in toluene(degassed, 850 mL) was added Grubbs II catalyst (2.02 g, 2.38 mmol) intoluene (degassed, 100 mL). The resulting mixture was stirred for 45 minat 85° C. The solvent was removed under reduced pressure and theresulting residue was purified by silica chromatography (0 to 40%gradient of EtOAc/heptane) to give tert-butyl1-oxo-8-azaspiro[4.5]dec-2-ene-8-carboxylate (20.76 g, 83 mmol) as brownsolid. A solution of this compound and DDQ (565 mg, 2.49 mmol) intoluene (540 mL) was stirred for 15 min at RT. The resulting bright redsolution was filtered through a pad of Celite. Charcoal (200 g) wasadded and the resulting suspension was stirred for 2 h at RT. Themixture was filtered through a pad of Celite and the volatiles wereremoved under reduce pressure. The resulting residue was purified bysilica chromatography (0 to 40% gradient of EtOAc/heptane) to givetert-butyl 1-oxo-8-azaspiro[4.5]dec-2-ene-8-carboxylate (15.6 g, 62.3mmol) as white solid. ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 7.63-7.74 (m,1H), 6.20 (dt, J=5.81, 2.15 Hz, 1H), 3.99-4.25 (m, 2 H), 2.92 (t,J=11.62 Hz, 2H), 2.63 (s, 2H), 1.72-1.86 (m, 2H), 1.49 (s, 9H), 1.29 (d,J=12.88 Hz, 2H).

Intermediate 25 tert-butyl1-(1,1-dimethylethylsulfinamido)-3-methyl-8-azaspiro[4.5]decane-8-carboxylate

Step a: To a suspension of tert-butyl1-oxo-8-azaspiro[4.5]dec-2-ene-8-carboxylate (4.2 g, 16.71 mmol) and CuI(6.37 g, 33.4 mmol) in Et₂O (100 mL) was added (at 0° C. and under N₂)MeLi (1.6 M in THF, 31.3 mL, 50.1 mmol). After stirring for 90 min at 0°C., the mixture was poured into a separation funnel containing sat. aqNH₄Cl and it was extracted with EtOAc (3×15 mL). The combined organicphases were dried over MgSO₄, filtered and the volatiles were removedunder reduced pressure. The resulting residue was purified by silicachromatography (0 to 50% gradient of EtOAc/heptane) to give tert-butyl3-methyl-1-oxo-8-azaspiro[4.5]dec-2-ene-8-carboxylate (4.23 g, 15.82mmol) as colorless oil. ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 3.89-4.00(m, 1H), 3.83 (d, J=13.39 Hz, 1H), 3.11 (ddd, J=13.64, 10.36, 3.28 Hz,1H), 2.99 (ddd, J=13.58, 10.42, 3.54 Hz, 1H), 2.47-2.59 (m, 1H),2.19-2.36 (m, 2H), 1.74-1.97 (m, 2H), 1.50-1.65 (m, 2H), 1.48 (s, 9H),1.33-1.44 (m, 2H), 1.17 (d, J=6.32 Hz, 3H).

Step b: A solution of tert-butyl3-methyl-1-oxo-8-azaspiro[4.5]dec-2-ene-8-carboxylate (502 mg, 1.878mmol), titanium(IV) ethoxide (1.57 mL, 7.51 mmol), and2-methylpropane-2-sulfinamide (455 mg, 3.76 mmol) in THF (12.5 mL) wasstirred for 16 h at 65° C. After cooling to 0° C., MeOH (3 mL) was addedfollowed by lithium borohydride (123 mg, 5.63 mmol). The resultingmixture was stirred at 0° C. for 1 h. sat. aq NH₄Cl was slowly added toquench the excess of borohydride followed by addition of EtOAc (30 mL).The resulting mixture was vigorously stirred for 15 min and thenfiltered through a pad of Celite. The volatiles were removed underreduced pressure and the resulting residue was purified by silicachromatography (0 to 75% gradient of EtOAc/heptane) to give tert-butyl1-(1,1-dimethylethylsulfinamido)-3-methyl-8-azaspiro[4.5]decane-8-carboxylate(463 mg, 1.243 mmol) as a white solid.

Intermediates 26a/b (1R,3S)-benzyl1-((R)-1,1-dimethylethylsulfinamido)-3-methyl-8-azaspiro[4.5]decane-8-carboxylate& (R,3R)-benzyl1-((R)-1,1-dimethylethylsulfinamido)-3-methyl-8-azaspiro[4.5]decane-8-carboxylate

Step a: A mixture of tert-butyl3-methyl-1-oxo-8-azaspiro[4.5]dec-2-ene-8-carboxylate (4.23 g, 15.82mmol) and TFA (17 mL) in DCM (80 mL) was stirred for 30 min at RT. Thevolatiles were removed under reduced pressure. A mixture of theresulting residue, DIPEA (13.82 mL, 79 mmol), and benzyl chloroformate(3.39 mL, 23.73 mmol) in DCM (80 mL) was stirred for 16 h at RT. Themixture was poured into a separation funnel containing sat. aq NH₄Cl andit was extracted with DCM (3×25 mL). The combined organic phases weredried over MgSO₄, filtered and the volatiles were removed under reducedpressure. The resulting residue was purified by silica chromatography (0to 40% gradient of EtOAc/heptane) to give benzyl3-methyl-1-oxo-8-azaspiro[4.5]decane-8-carboxylate (4.58 g, 15.20 mmol)as light yellow oil. MS m/z 302.2 (M+H)⁺.

Step b: Benzyl 3-methyl-1-oxo-8-azaspiro[4.5]decane-8-carboxylate (4.58g, 15.20 mmol) was further purified by chiral SFC as follows: column: IA21×250 mm, flow rate: 70 g per minute, mobile phase: 45% (9:1 EtOH:MeCN)in CO₂, detection: 220 nm UV to give (R)-benzyl3-methyl-1-oxo-8-azaspiro[4.5]decane-8-carboxylate (2.02 g, 6.70 mmol),R_(t): 2.0 min; and (S)-benzyl3-methyl-1-oxo-8-azaspiro[4.5]decane-8-carboxylate (2.11 g, 7.0 mmol),R_(t): 3.6 min.

Step c: A solution of (R)-benzyl3-methyl-1-oxo-8-azaspiro[4.5]decane-8-carboxylate (2.02 g, 6.70 mmol),titanium(IV) ethoxide (5.62 mL, 26.8 mmol), and(R)-2-methylpropane-2-sulfinamide (1.625 g, 13.4 mmol) in THF (67 mL)was stirred for 16 h at 65° C. After cooling to −78° C., MeOH (12 mL)was added followed by lithium borohydride (0.438 g, 20.11 mmol). Theresulting mixture was stirred for 16 h at −78° C. to RT. Sat. aq NH₄Clwas slowly added to quench the excess of borohydride followed byaddition of EtOAc (100 mL). The resulting mixture was vigorously stirredfor 15 min and then filtered through a pad of Celite. The volatiles wereremoved under reduced pressure and the resulting residue was purified bysilica chromatography (5 to 90% gradient of EtOAc/heptane) to give(1R,3R)-benzyl1-((R)-1,1-dimethylethylsulfinamido)-3-methyl-8-azaspiro[4.5]decane-8-carboxylate(1.94 g, 4.77 mmol) as a white solid. MS m/z 407.3 (M+H)⁺.

Step c (from enantiomer): The same procedure was followed starting from(S)-benzyl 3-methyl-1-oxo-8-azaspiro[4.5]decane-8-carboxylate to give(1R,3S)-benzyl1-((R)-1,1-dimethylethylsulfinamido)-3-methyl-8-azaspiro[4.5]decane-8-carboxylate.

Intermediate 27 (1R,3R)-tert-butyl3-((tert-butyldimethylsilyl)oxy)-1-((R)-1,1-dimethylethylsulfinamido)-8-azaspiro[4.5]decane-8-carboxylate)

Step a: A mixture of CuCl (142 mg, 1.432 mmol), (S)-TolBINAP (972 mg,1.432 mmol), and sodium tert-butoxide (138 mg, 1.432 mmol) in THF (60mL) was stirred for 30 min at RT. B₂pin₂ (13.34 g, 52.5 mmol) in THF (20mL) was added and the resulting mixture was stirred for 10 min at RT.Tert-butyl 1-oxo-8-azaspiro[4.5]dec-2-ene-8-carboxylate (12.0 g, 47.7mmol) in THF (50 mL) was added followed by MeOH (3.9 mL, 95 mmol). Theresulting mixture was stirred for 16 h at RT. H₂O (150 mL) was addedfollowed by sodium perborate (36.7 g, 239 mmol) and the resultingmixture was vigorously stirred for 1 h at RT. The resulting greensuspension was filtered through a pad of Celite, poured into aseparation funnel containing sat. aq NaHCO₃: sat. aq Na₂SO₃ (1:1, 300mL) and extracted with EtOAc (4×40 mL). The combined organic phases weredried over MgSO₄, filtered, and the volatiles were removed under reducedpressure to give (R)-tert-butyl3-hydroxy-1-oxo-8-azaspiro[4.5]decane-8-carboxylate crude. Enantiomericdetermination of this mixture show 90% ee (R_(t)(S): 1.59 min, R_(t)(R):1.80 min; chiral SFC; column: IA 4.6×100 mm, flow rate: 70 g per minute,mobile phase: 5-55% MeOH in CO₂, detection: 220 nm UV).

A mixture of (R)-tert-butyl3-hydroxy-1-oxo-8-azaspiro[4.5]decane-8-carboxylate crude (theor 47.7mmol), imidazole (4.87 g, 71.6 mmol), and TBSCl (8.99 g, 59.6 mmol) inDMF (120 mL) was stirred for 16 h at RT. The reaction mixture was pouredinto a separation funnel containing sat. aq NH₄C1:H₂O (1:1, 250 mL) andit was extracted with Et₂O (5×50 mL). The combined organic phases weredried over MgSO₄, filtered and the volatiles were removed under reducedpressure. The resulting residue was purified by silica chromatography (0to 30% gradient of EtOAc/heptane) to give (R)-tert-butyl3-((tert-butyldimethylsilyl)oxy)-1-oxo-8-azaspiro[4.5]decane-8-carboxylate(13.115 g, 34.2 mmol) as colorles oil that solidify upon standing.

Step b: A solution of (R)-tert-butyl3-((tert-butyldimethylsilyl)oxy)-1-oxo-8-azaspiro[4.5]decane-8-carboxylate(8 g, 20.86 mmol), titanium(IV) ethoxide (17.49 mL, 83.0 mmol), and(R)-2-methylpropane-2-sulfinamide (5.06 g, 41.7 mmol) in THF (100 mL)was stirred for 16 h at 65° C. After cooling to −78° C., MeOH (15 mL)was added followed by lithium borohydride (1.363 g, 62.6 mmol). Theresulting mixture was stirred for 16 h at −78° C. Sat. aq NH₄Cl wasslowly added to quench the excess of borohydride followed by addition ofEtOAc (100 mL). The resulting mixture was vigorously stirred for 15 minand then filtered through a pad of Celite. The volatiles were removedunder reduced pressure and the resulting residue was purified by silicachromatography (0 to 50% gradient of EtOAc/heptane) to give(1R,3R)-tert-butyl3-((tert-butyldimethylsilyl)oxy)-1-((R)-1,1-dimethylethylsulfinamido)-8-azaspiro[4.5]decane-8-carboxylate(5.3 g, 10.84 mmol) as a white solid. MS m/z 489.3 (M+H)⁺.

Intermediate 28 (1R,3S)-tert-butyl1-((R)-1,1-dimethylethylsulfinamido)-3-hydroxy-8-azaspiro[4.5]decane-8-carboxylate)

Step a: A mixture of (1R,3R)-tert-butyl3-((tert-butyldimethylsilyl)oxy)-1-((R)-1,1-dimethylethylsulfinamido)-8-azaspiro[4.5]decane-8-carboxylate(3.84 g, 7.86 mmol) and TBAF (1 M in THF; 8.64 mL, 8.64 mmol) in THF (40mL) was stirred for 30 min at RT. The volatiles were removed underreduced pressure and the resulting residue was purified by silicachromatography (0 to 10% gradient of MeOH/DCM) to give (1R,3R)-tert-butyl1-((R)-1,1-dimethylethylsulfinamido)-3-hydroxy-8-azaspiro[4.5]decane-8-carboxylate(2.94 g, 7.86 mmol). MS m/z 375.3 (M+H)⁺.

Step b: To a solution of (1R,3R)-tert-butyl1-((R)-1,1-dimethylethylsulfinamido)-3-hydroxy-8-azaspiro[4.5]decane-8-carboxylate(3.0 g, 8.01 mmol), triphenylphosphine (4.2 g, 16.02 mmol), andisoquinoline-1-carboxylic acid (4.16 g, 24.03 mmol) in THF (80 mL) wasadded DIAD (3.1 mL, 16.02 mmol). The resulting mixture was stirred for 1h at RT. The reaction was diluted with EtOAc (50 mL), filtered through apad of Celite, poured into a separation funnel containing sat. aq NaHCO₃and extracted with EtOAc (3×25 mL). The combined organic phases weredried over MgSO₄, filtered and the volatiles were removed under reducedpressure. The resulting residue was purified by silica chromatography (0to 4% gradient of MeOH/DCM) to give(2S,4R)-8-(tert-butoxycarbonyl)-4-((R)-1,1-dimethylethylsulfinamido)-8-azaspiro[4.5]decan-2-ylisoquinoline-1-carboxylate (3.65 g, 6.89 mmol) as orange solid. MS m/z530.3 (M+H)⁺.

Step c: A mixture of(2S,4R)-8-(tert-butoxycarbonyl)-4-((R)-1,1-dimethylethylsulfinamido)-8-azaspiro[4.5]decan-2-ylisoquinoline-1-carboxylate (3.65 g, 6.89 mmol) and lithium hydroxide(2.95 g, 68.9 mmol) in THF:H₂O (1:1, 70 mL) was stirred for 2 h at RT.The mixture was poured into a separation funnel containing sat. aq NH₄Cland it was extracted with EtOAc (3×15 mL). The combined organic phaseswere dried over MgSO₄, filtered and the volatiles were removed underreduced pressure. The resulting residue was purified by silicachromatography (0 to 10% gradient of MeOH/DCM) to give(1R,3S)-tert-butyl1-((R)-1,1-dimethylethylsulfinamido)-3-hydroxy-8-azaspiro[4.5]decane-8-carboxylate(2.35 g, 6.27 mmol) as white solid. MS m/z 275.2 (M+H-Boc)⁺.

Intermediate 29 (1R,3S)-tert-butyl1-((R)-1,1-dimethylethylsulfinamido)-3-(isobutyryloxy)-8-azaspiro[4.5]decane-8-carboxylate)

To a solution of (1R,3R)-tert-butyl1-((R)-1,1-dimethylethylsulfinamido)-3-hydroxy-8-azaspiro[4.5]decane-8-carboxylate(200 mg, 0.534 mmol), triphenylphosphine (280 mg, 1.068 mmol), andisobutyric acid (146 μL, 1.602 mmol) in THF (5 mL) was added DIAD (208μL, 1.068 mmol). The resulting mixture was stirred for 16 h at RT. Thereaction was diluted with EtOAc (50 mL), filtered through a pad ofCelite, poured into a separation funnel containing sat. aq NaHCO₃, andextracted with EtOAc (3×10 mL). The combined organic phases were driedover MgSO₄, filtered, and the volatiles were removed under reducedpressure. The resulting residue was purified by silica chromatography (0to 7% gradient of MeOH/DCM) to give (1R,3S)-tert-butyl1-((R)-1,1-dimethylethylsulfinamido)-3-(isobutyryloxy)-8-azaspiro[4.5]decane-8-carboxylate)(237 mg, 0.534 mmol). MS m/z 345.3 (M+H-Boc)⁺.

Intermediates 30a/b/c (1R,3R)-tert-butyl1-((R)—N,2-dimethylpropan-2-ylsulfinamido)-3-hydroxy-8-azaspiro[4.5]decane-8-carboxylate,(1R,3R)-tert-butyl1-((R)-1,1-dimethylethylsulfinamido)-3-methoxy-8-azaspiro[4.5]decane-8-carboxylate,& (1R,3R)-tert-butyl1-((R)—N,2-dimethylpropan-2-ylsulfinamido)-3-methoxy-8-azaspiro[4.5]decane-8-carboxylate

A mixture of (1R,3R)-tert-butyl1-((R)-1,1-dimethylethylsulfinamido)-3-hydroxy-8-azaspiro[4.5]decane-8-carboxylate(142 mg, 0.378 mmol) and NaH (60% dispersion in mineral oil, 19 mg,0.473 mmol) in THF was stirred for 20 min at 0° C. Iodomethane (47 μL,0.756 mmol) was added and the resulting mixture was stirred for 4 h atRT. The volatiles were removed under reduced pressure and the resultingresidue was purified by HPLC (gradient elution 25-50% acetonitrile inwater, 5 mM NH₄OH modifier) to give (1R,3R)-tert-butyl1-((R)—N,2-dimethylpropan-2-ylsulfinamido)-3-hydroxy-8-azaspiro[4.5]decane-8-carboxylate(15.0 mg, 0.039 mmol). MS m/z 289.2 (M+H-Boc)⁺; (1R,3R)-tert-butyl1-((R)-1,1-dimethylethylsulfinamido)-3-methoxy-8-azaspiro[4.5]decane-8-carboxylate.MS m/z 289.2 (M+H-Boc)⁺; and (1R,3R)-tert-butyl1-((R)—N,2-dimethylpropan-2-ylsulfinamido)-3-methoxy-8-azaspiro[4.5]decane-8-carboxylate.MS m/z 303.2 (M+H-Boc)⁺.

Intermediate 31 (1R,3S)-tert-butyl1-((R)-1,1-dimethylethylsulfinamido)-3-methoxy-8-azaspiro[4.5]decane-8-carboxylate

A mixture of (1R,3S)-tert-butyl1-((R)-1,1-dimethylethylsulfinamido)-3-hydroxy-8-azaspiro[4.5]decane-8-carboxylate(500 mg, 1.335 mmol), silver (I) oxide (340 mg, 1.468 mmol), andiodomethane (250 μL, 4.0 mmol) in DCM (5 mL) was stirred (protected fromthe light) for 24 h at RT and 24 h at 45° C. After cooling to RT, themixture was filtered through a pad of Celite, the volatiles were removedunder reduced pressure, and the resulting residue was purified by silicachromatography (0 to 5% gradient of MeOH/DCM) to give (1R,3S)-tert-butyl1-((R)-1,1-dimethylethylsulfinamido)-3-methoxy-8-azaspiro[4.5]decane-8-carboxylate(248 mg, 0.638 mmol). MS m/z 289.2 (M+H-Boc)⁺.

Intermediate 32 racemic tert-butyl1-((tert-butoxycarbonyl)amino)-3,3-difluoro-8-azaspiro[4.5]decane-8-carboxylate

Step a: A mixture of tert-butyl3-((tert-butyldimethylsilyl)oxy)-1-(1,1-dimethylethylsulfinamido)-8-azaspiro[4.5]decane-8-carboxylate(365 mg, 0.746 mmol) and HCl (4 M in dioxane, 1.86 mL, 7.46 mmol) inMeOH (4 mL) was stirred for 1 h at 40° C. After cooling to RT, thevolatiles were removed under reduced pressure to give a white solid. MSm/z 171.1 (M+H)⁺. A mixture of this residue, DIPEA (2.6 mL, 14.92 mmol),and Boc₂O (407 mg, 1.865 mmol) in THF (15 mL) was stirred for 16 h atRT. The mixture was poured into a separation funnel containing sat. aqNH₄Cl and it was extracted with Et₂O (5×10 mL). The combined organicphases were dried over MgSO₄, filtered and the volatiles were removedunder reduced pressure. The resulting residue was purified by silicachromatography (10 to 80% gradient of EtOAc/heptane) to give tert-butyl1-((tert-butoxycarbonyl)amino)-3-hydroxy-8-azaspiro[4.5]decane-8-carboxylate(275 mg, 0.742 mmol). MS m/z 271.3 (M+H-Boc)⁺.

Step b: A mixture of tert-butyl1-((tert-butoxycarbonyl)amino)-3-hydroxy-8-azaspiro[4.5]decane-8-carboxylate(275 mg, 0.742 mmol) and Dess-Martin periodinane (472 mg, 1.113 mmol) inDCM (7.5 mL) was stirred for 2 h at 0° C. The mixture was poured into aseparation funnel containing sat. aq NaHCO₃ and it was extracted withDCM (3×10 mL). The combined organic phases were dried over MgSO₄,filtered and the volatiles were removed under reduced pressure. Theresulting residue was purified by silica chromatography (5 to 75%gradient of EtOAc/heptane) to give tert-butyl1-((tert-butoxycarbonyl)amino)-3-oxo-8-azaspiro[4.5]decane-8-carboxylate(135 mg, 0.366 mmol). ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 4.57 (d,J=9.09 Hz, 1H), 4.16 (d, J=8.08 Hz, 1H), 3.89-4.08 (m, 2H), 2.77-2.93(m, 2 H), 2.71 (dd, J=18.95, 8.08 Hz, 1H), 2.50 (d, J=18.19 Hz, 1H),2.07-2.24 (m, 2H), 1.76 (td, J=12.82, 4.67 Hz, 1H), 1.58-1.70 (m, 1H),1.42-1.53 (m, 18H), 1.25-1.38 (m, 1H).

Step c: A mixture of tert-butyl1-((tert-butoxycarbonyl)amino)-3-oxo-8-azaspiro[4.5]decane-8-carboxylate(95 mg, 0.258 mmol) and DeoxoFluor (190 μL, 1.031 mmol) in DCM (1 mL)was stirred for 48 h at 50° C. The mixture was poured into a separationfunnel containing sat. aq NaHCO₃/ice and it was extracted with EtOAc(3×5 mL). The combined organic phases were dried over MgSO₄, filteredand the volatiles were removed under reduced pressure. The resultingresidue was purified by silica chromatography (0 to 30% gradient ofEtOAc/heptane) to give tert-butyl1-((tert-butoxycarbonyl)amino)-3,3-difluoro-8-azaspiro[4.5]decane-8-carboxylate(52 mg, 0.133 mmol). ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 4.55 (d,J=9.35 Hz, 1H), 3.78-4.02 (m, 3H), 2.64-2.86 (m, 2H), 2.38-2.59 (m, 1H),2.10-2.32 (m, 1H), 1.79-2.10 (m, 2H), 1.58 (qd, J=12.72, 3.79 Hz, 1H),1.27-1.52 (m, 21H).

The following compounds were synthesized using the above procedure ormodifications to the above procedure using the chirally pure(1R,3R)-tert-butyl3-((tert-butyldimethylsilyl)oxy)-1-((R)-1,1-dimethylethylsulfinamido)-8-azaspiro[4.5]decane-8-carboxylateas starting material.

TABLE 2

Intermediate 33 1-amino-2,8-diazaspiro[4.5]dec-1-en-3-one

Step a: A solution of diisopropylamine (0.320 mL, 2.245 mmol) in THF (4mL) was cooled to −78° C. and treated with n-butyllithium (1.3 mL, 2.080mmol) then stirred for 5 min at −78° C. and warmed to 0° C. giving asolution of LDA to be used subsequently. To a −78° C. solution oftert-butyl 4-cyanopiperidine-1-carboxylate (153 mg, 0.728 mmol) in THF(10 mL) was added the prepared solution of LDA (2.8 mL) dropwise and theresulting mixture stirred for 10 min at −78° C., then for 10 min at −10°C. Reaction was recooled to −78° C. and a solution of allyl-Br (80 μL,0.924 mmol) in THF (2 mL) was added dropwise. The resulting reactionmixture was stirred for 1 h at RT and the volatiles were removed underreduced pressure. The aqueous was extracted with EtOAc, the combinedorganic phases were washed with water, brine, dried over Na₂SO₄,filtered and concentrated under reduced pressure. The residue waspurified by silica chromatography (0 to 50% gradient of EtOAc/heptane)to give tert-butyl 4-allyl-4-cyanopiperidine-1-carboxylate (40 mg, 0.16mmol) as a colorless oil. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 5.99-5.70 (m,1H), 5.23 (q, J=1.1 Hz, 1H), 5.20 (dtd, J=3.3, 2.1, 1.1 Hz, 1H), 3.96(d, J=13.7 Hz, 2H), 2.86 (s, 2H), 2.36 (dt, J=7.5, 1.3 Hz, 2H), 1.84(dq, J=13.7, 2.6 Hz, 2H), 1.40 (s, 11H).

Step b: A solution of tert-butyl 4-allyl-4-cyanopiperidine-1-carboxylate(22 mg, 0.088 mmol) in DCM (1.5 mL) and NaOH (2.5 M in MeOH, 0.176 mL,0.439 mmol) was aerated with ozone (diffusing aerator) at −78° C. for 30minutes. The reaction was purged with oxygen then partitioned betweenwater and DCM. The phases were separated, the organic collected and theaqueous extracted with DCM (2×5 mL). The combined organic phases wereconcentrated under reduce pressure. The resulting residue was taken upin MeOH and stirred for 24 h at 65° C. After cooling to RT, thevolatiles were removed under reduced pressure and the resulting residuewas purified by silica chromatography (0 to 70% gradient ofEtOAc/heptane) to give tert-butyl4-cyano-4-(2-methoxy-2-oxoethyl)piperidine-1-carboxylate (21 mg, 0.074mmol) as a colorless oil. ¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 4.14 (s,2H), 3.75 (s, 3H), 3.08 (t, J=12.9 Hz, 2H), 2.62 (s, 2H), 2.15-2.02 (m,2H), 1.59-1.48 (m, 2H), 1.46 (s, 9H). TLC (50% EtOAc/heptane (stainedw/KMnO4), R_(f)=0.5).

Step c: A solution of tert-butyl4-cyano-4-(2-methoxy-2-oxoethyl)piperidine-1-carboxylate (287 mg, 1.017mmol) and NH₃ (7 N in MeOH, 3.0 mL, 21.00 mmol) in MeOH (5 mL) wasstirred in a sealed tube for 48 h at 120° C. After cooling to RT, thevolatiles were removed under reduced pressure to give a white solid. Thesolid was triturated with EtOAc and filtered to give tert-butyl1-amino-3-oxo-2,8-diazaspiro[4.5]dec-1-ene-8-carboxylate (157 mg, 0.587mmol) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 8.44 (s, 1H),8.02 (s, 1H), 3.98 (d, J=13.3 Hz, 2H), 2.71 (s, 2H), 2.34 (s, 2H), 1.81(td, J=12.9, 4.6 Hz, 2H), 1.49-1.30 (m, 11H). MS m/z 268 (M+H)⁺.

Intermediates 34a/b racemic tert-butyl2-fluoro-1-oxo-8-azaspiro[4.5]decane-8-carboxylate & tert-butyl2,2-difluoro-1-oxo-8-azaspiro[4.5]decane-8-carboxylate

To a −78° C. solution of NaHMDS (1 M in THF, 8.68 mL, 8.68 mmol) wasadded a solution of tert-butyl 1-oxo-8-azaspiro[4.5]decane-8-carboxylate(2.0 g, 7.89 mmol) in THF (5 mL). After stirring for 30 min at thistemperature, a solution of N-fluorobenzenesulfonamide (2.49 g, 7.89mmol) in THF (10 mL) was added. After 3 h stirring at −78° C., it wasdiluted with sat. aq NaHCO₃ (100 mL) and extracted with DCM (3×100 mL).The combined organic phases were washed with brine, dried over Na₂SO₄,filtered, and concentrated under reduced pressure. The resulting residuewas purified by silica chromatography (0 to 25% gradient ofEtOAc/heptane) to give racemic tert-butyl2-fluoro-1-oxo-8-azaspiro[4.5]decane-8-carboxylate (351 mg, 1.29 mmol).MS m/z 272.1 (M+H)⁺ and difluoro ketone which coelutes with startingmaterial. The combined coeluted fractions of difluoro ketone/startingwere repurified by silica chromatography (0 to 5% gradient of MeOH/DCM)to give tert-butyl2,2-difluoro-1-oxo-8-azaspiro[4.5]decane-8-carboxylate (573 mg, 1.98mmol). MS m/z 290.1 (M+H)⁺.

Intermediate 35 (S)-tert-butyl4-((R)-1,1-dimethylethylsulfinamido)-2-oxa-8-azaspiro[4.5]decane-8-carboxylate

Step a: A solution of tert-butyl4-hydroxy-2-oxa-8-azaspiro[4.5]decane-8-carboxylate (544 mg, 2.11 mmol)and Dess-Martin periodinane (1.39 g, 3.17 mmol) in DCM (10 mL) wasstirred for 2 h at 0° C. Sat. aqNaHCO₃:sat. aqNa₂S₂O₃ (1:1, 10 mL) wasadded, the organic phase was separated and the aqueous phase wasextracted with DCM (3×10 mL). The combined organic phases were driedover Na₂SO₄, filtered and the volatiles were removed under reducedpressure. The resulting residue was purified by silica chromatography (0to 50% gradient of EtOAc/heptane) to give tert-butyl4-oxo-2-oxa-8-azaspiro[4.5]decane-8-carboxylate (470 mg, 1.84 mmol) as acolorless oil which crystallized upon standing. ¹H NMR (400 MHz,CHLOROFORM-d) δ ppm 4.08 (s, 2H), 4.05 (s, 2H), 3.88 (dt, J=13.7, 4.9Hz, 2H), 3.12 (ddd, J=13.6, 9.8, 3.6 Hz, 2H), 1.75 (ddd, J=13.9, 9.7,4.2 Hz, 2H), 1.58-1.51 (m, 2H), 1.48 (s, 9H). MS m/z 256.2 (M+H)⁺.

Step b: A solution of tert-butyl4-oxo-2-oxa-8-azaspiro[4.5]decane-8-carboxylate (220 mg, 0.86 mmol),titanium(IV) ethoxide (725 μL, 3.45 mmol), and(R)-2-methylpropane-2-sulfinamide (209 mg, 1.72 mmol) in THF (4 mL) wasstirred for 1 h at 90° C. After cooling to 0° C., lithium borohydride(23 mg, 1.06 mmol) was added. After stirring for 30 min, the reactionmixture was quenched by addition of MeOH. The volatiles were removedunder reduce pressure. The resulting residue was diluted with brine andit was extracted with EtOAc (4×10 mL). The combined organic phases weredried over Na₂SO₄, filtered, the volatiles were removed under reducedpressure, and the resulting residue was purified by silicachromatography (0 to 100% gradient of EtOAc/heptane) to give(S)-tert-butyl4-((R)-1,1-dimethylethylsulfinamido)-2-oxa-8-azaspiro[4.5]decane-8-carboxylate(170 mg, 0.47 mmol). MS m/z 361.1 (M+H)⁺.

The following compounds were synthesized using the above procedure ormodifications to the above procedure using the corresponding ketone andsulfonamide.

TABLE 3

Intermediate 36 (1R)-tert-butyl1-((R)-1,1-dimethylethylsulfinamido)-2-methyl-8-azaspiro[4.5]decane-8-carboxylate

Step a: To a solution of tert-butyl1-oxo-8-azaspiro[4.5]decane-8-carboxylate (2.2 g, 8.68 mmol) in THF (24mL) was added LiHMDS (1 M in THF, 8.68 mL, 8.68 mmol) at 0-5° C. Afterstirring the mixture for 30 min at this temperature, iodomethane (0.543mL, 8.68 mmol) was added. The resulting mixture was allowed to warm toRT and stirred for 2 h. The reaction mixture was diluted with EtOAc andquenched with sat. aq NaHCO₃. The organic phase was washed with brine,dried over Na₂SO₄, filtered and concentrated under reduced pressure. Theresulting brown oil was purified by silica chromatography (0 to 25%gradient of EtOAc/heptane) to give tert-butyl2-methyl-1-oxo-8-azaspiro[4.5]decane-8-carboxylate (1.3 g, 4.86 mmol).MS m/z 268.1. (M+H)⁺.

Step b: A solution of tert-butyl2-methyl-1-oxo-8-azaspiro[4.5]decane-8-carboxylate (267 mg, 0.999 mmol),titanium(IV) ethoxide (837 μL, 3.99 mmol), and(R)-2-methylpropane-2-sulfinamide (242 mg, 1.997 mmol) in THF (10 mL)was stirred for 24 h at 85° C. After cooling to −78° C., MeOH (12 mL)was added followed by lithium borohydride (65.3 mg, 3.00 mmol). Theresulting mixture was stirred at −78° C. to RT for 16 h. Sat. aq NH₄Clwas slowly added to quench the excess of borohydride followed byaddition of EtOAc (100 mL). The resulting mixture was vigorously stirredfor 15 min and then filtered through a pad of Celite. The volatiles wereremoved under reduced pressure and the resulting residue was purified bysilica chromatography (0 to 60% gradient of EtOAc/heptane (containing0.25% of Et₃N)) to give (1R)-tert-butyl1-((R)-1,1-dimethylethylsulfinamido)-2-methyl-8-azaspiro[4.5]decane-8-carboxylate(92 mg, 0.247 mmol). MS m/z 373.1 (M+H)⁺.

Intermediates 37a/b (3S,4S)-tert-butyl4-((R)-1,1-dimethylethylsulfinamido)-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-carboxylate& (3R,4S)-tert-butyl4-((R)-1,1-dimethylethylsulfinamido)-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-carboxylate

Step a: To a solution of of tert-butyl4-oxo-2-oxa-8-azaspiro[4.5]decane-8-(2.47 g, 9.67 mmol) in THF (24 mL)was added LiHMDS (1 M in THF, 9.67 mL, 9.67 mmol) at −78° C. Afterstirring the mixture for 30 min at this temperature, iodomethane (0.605mL, 9.67 mmol) in THF (10 mL) was added. The resulting mixture wasallowed to warm to RT and stirred for 1 h. The reaction mixture wasdiluted with EtOAc and quenched with sat. aq NaHCO₃. The organic phasewas washed with brine, dried over Na₂SO₄, filtered, and concentratedunder reduced pressure. The resulting brown oil was purified by silicachromatography (0 to 20% gradient of EtOAc/heptane) to give tert-butyl3-methyl-4-oxo-2-oxa-8-azaspiro[4.5]decane-8-carboxylate (318 mg, 1.181mmol). MS m/z 270.2. (M+H)⁺.

Step b: A solution of tert-butyl3-methyl-4-oxo-2-oxa-8-azaspiro[4.5]decane-8-carboxylate (318 mg, 1.181mmol), titanium(IV) ethoxide (990 μL, 4.72 mmol), and(R)-2-methylpropane-2-sulfinamide (286 mg, 2.361 mmol) in THF (4 mL) wasstirred for 90 min at 90° C. After cooling to 0° C., lithium borohydride(65.3 mg, 3.00 mmol) was added in one portion and the resulting mixturewas stirred stirred for 16 h at RT. Sat. aq NH₄Cl was slowly added toquench the excess of borohydride followed by addition of EtOAc (25 mL).The resulting mixture was vigorously stirred for 15 min and thenfiltered through a pad of Celite. The organic phase was washed with sat.aq NaHCO₃, brine, dried over Na₂SO₄, filtered, and the volatiles wereremoved under reduced pressure. The resulting residue was purified bysilica chromatography (0 to 100% gradient of EtOAc/heptane) to give(4S)-tert-butyl4-((R)-1,1-dimethylethylsulfinamido)-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-carboxylate(88 mg, 0.235 mmol). MS m/z 375.2 (M+H)⁺.

Step c: The diastereomers were separated by chiral SFC as follows:column: LUXC4 30×250 mm, flow rate: 80 g per minute, mobil phase: 20%MeOH in CO₂, detection: 210 nm to give (3R,4S)-tert-butyl4-((R)-1,1-dimethylethylsulfinamido)-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-carboxylate,R_(t)=4.0 min; and (3S,4S)-tert-butyl4-((R)-1,1-dimethylethylsulfinamido)-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-carboxylate,R_(t)=4.55 min.

Intermediate 38 (3S,4S)-tert-butyl4-((R)-1,1-dimethylethylsulfinamido)-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-carboxylate

Step a: To a −10° C. solution of diisopropylamine (23.4 mL, 166 mmol) inTHF (220 mL) was added nBuLi (2.5 M in hexane, 64.1 mL, 160 mmol)dropwise. After stirring for 30 min at this temperature, 1-tert-butyl4-ethyl piperidine-1,4-dicarboxylate (27.5 g, 107 mmol) in THF (50 mL)was added dropwise and the resulting mixture was stirred for 30 min at0° C. (S)-2-((tert-butyldimethylsilyl)oxy)propanal (20.47 mL, 102 mmol)was added and the mixture was stirred for 1 h at 0° C. and 1 h at RT.The reaction was diluted with sat. aqNaHCO₃:H₂O (1:4, 125 mL), EtOAc (50mL) was added, and the phases were separated. The aqueous phase wasfurther extracted with EtOAc (3×100 mL). The combined organic phaseswere dried over Na₂SO₄, filtered, and the solvent was removed underreduced pressure. The resulting residue was used in next step withoutfurther purification. MS m/z 346.4 (M+H-Boc)⁺.

Step b: To a solution of crude 1-tert-butyl 4-ethyl4-((2S)-2-((tert-butyldimethylsilyl)oxy)-1-hydroxypropyl)piperidine-1,4-dicarboxylate(95 g, 214 mmol) in THF (600 mL) was added portionwise LiBH₄ (7.0 g, 321mmol) and the resulting mixture was stirred for 16 h at RT. Aftercooling to 0° C., sat. aq NaHCO₃:H₂O (1:2, 150 mL) was added and theresulting mixture was vigorously stirred until no more bubbling wasobserved. EtOAc (100 mL) was added, the mixture was filtered, the phaseswere separated, and the aqueous phase was further extracted with EtOAc(3×50 mL). The combined organic phases were washed with brine, driedover Na₂SO₄, filtered, and the volatiles were removed under reducedpressure to give tert-butyl4-((2S)-2-((tert-butyldimethylsilyl)oxy)-1-hydroxypropyl)-4-(2-hydroxyethyl)piperidine-1-carboxylate(64.8 g, 161 mmol) which was used in next step without furtherpurification.

Step c: A solution of tert-butyl4-((2S)-2-((tert-butyldimethylsilyl)oxy)-1-hydroxypropyl)-4-(2-hydroxyethyl)piperidine-1-carboxylate(64.8 g, 161 mmol) and TBAF (1 M in THF, 242 mL, 242 mmol) in THF (500mL) was stirred for 2 h at RT. Sat. aq NaHCO₃:H₂O (1:2, 150 mL) wereadded, the phases were separated, and the aqueous phase was furtherextracted with EtOAc (3×100 mL). The combined organic phases were washedwith brine, dried over Na₂SO₄, filtered, and the volatiles were removedunder reduced pressure. The resulting residue was purified by silicachromatography (20 to 100% gradient of EtOAc/heptane) to give tert-butyl4-((2S)-1,2-dihydroxypropyl)-4-(2-hydroxyethyl)piperidine-1-carboxylate(39.25 g, 136 mmol) as a semi-solid colorless oil.

Step d: To a 0° C. suspension of NaH (10.60 g, 424 mmol) in THF (600 mL)was added dropwise a solution of tert-butyl4-((2S)-1,2-dihydroxypropyl)-4-(2-hydroxyethyl)piperidine-1-carboxylate(35.06 g, 121 mmol) and TsCl (23.10 g, 121 mmol) in THF (200 mL). Theresulting mixture was stirred for 1 h at 0° C. Sat. aq NH₄Cl (˜5 mL) wasadded slowly at −20° C. and the reaction was vigorously stirred until nomore bubbling was observed. At this point, sat. aq NH₄Cl (100 mL) wasadded followed by brine (100 mL) and the mixture was extracted withEtOAc (3×100 mL). The combined organic phases were dried over Na₂SO₄,filtered, and the solvent was removed under reduced pressure to give(3S)-tert-butyl4-hydroxy-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-carboxylate (32.19 g,119 mmol) which was used in next step without further purification. MSm/z 171.1 (M-Boc)⁻.

Step e: A solution of (3S)-tert-butyl4-hydroxy-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-carboxylate (32.19 g,119 mmol) and Dess-Martin periodinane (67.4 g, 154 mmol) in DCM (300 mL)was stirred for 2 h at 0° C. After warming to RT, the volatiles wereremoved under reduced pressure and the resulting residue was purified bysilica chromatography (0 to 40% gradient of EtOAc/heptane) to give(S)-tert-butyl 3-methyl-4-oxo-2-oxa-8-azaspiro[4.5]decane-8-carboxylate(27.68 g, 92 mmol) as a pale yellow oil. ¹H NMR (400 MHz, CHLOROFORM-d)δ ppm 4.09 (d, J=9.60 Hz, 1H), 3.66-3.86 (m, 4H), 3.03 (ddd, J=13.77,9.73, 3.79 Hz, 1H), 2.90 (ddd, J=13.64, 10.23, 3.41 Hz, 1H), 1.68 (ddd,J=13.83, 9.92, 4.29 Hz, 1H), 1.41-1.59 (m, 2H), 1.30-1.40 (m, 10H),1.20-1.25 (m, 3H).

Step f: A solution of (3S)-tert-butyl3-methyl-4-oxo-2-oxa-8-azaspiro[4.5]decane-8-carboxylate (22.52 g mg, 84mmol), titanium(IV) ethoxide (70.1 mL, 334 mmol), and(R)-2-methylpropane-2-sulfinamide (21 g, 173 mmol) in THF (300 mL) wasstirred for 21 h at 90° C. After cooling to −4° C., MeOH (30 mL) wasadded, followed by dropwise addition (maintaining reaction temperaturebelow 2° C.) of lithium borohydride (1.82 g, 84 mmol) and the resultingmixture was stirred for 1 h at −4° C. Sat. aq NH₄Cl was slowly added toquench the excess of borohydride (gelatin-type formed) followed byaddition of EtOAc (500 mL). The resulting mixture was vigorously stirredfor 15 min at RT and then filtered through a pad of Celite followed byEtOAc (500 mL) wash. The volatiles were removed under reduced pressureand the resulting residue was purified by silica chromatography (0 to100% gradient of EtOAc/heptane) to give (3S,4S)-tert-butyl4-((R)-1,1-dimethylethylsulfinamido)-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-carboxylateas a 95:5 diastereomeric mixture (minor diastereomer (3R,4S)-tert-butyl4-((R)-1,1-dimethylethylsulfinamido)-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-carboxylate).

Step g: The diastereomers were separated by chiral SFC as follows:column: LC-4 30×250 mm, flow rate: 100 g per minute, mobil phase: 30%MeOH in CO₂, detection: 225 nm, R_(t): 0.95 min (minor diastereomerR_(t): 0.55 min) to give (3S,4S)-tert-butyl4-((R)-1,1-dimethylethylsulfinamido)-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-carboxylate(19 g, 50.68 mmol). MS m/z 375.2.

Intermediate 39 (4R)-4-amino-2-methyl-8-azaspiro[4.5]decan-2-ol

Step a: A mixture of (2R,4R)-4-amino-8-azaspiro[4.5]decan-2-oldihydrochloride salt (623 mg, 2.56 mmol), Na₂CO₃ (1357 mg, 12.80 mmol),and CbzCl (1048 mg, 6.14 mmol) in H₂O (5 mL) was stirred vigorously for30 min at RT. THF (0.5 mL) was added and the resulting mixture wasstirred for 18 h at RT. The mixture was diluted with water and DCM. Theseparated aqueous phase was extracted with DCM (2×10 mL). The combinedorganic phases were dried over Na₂SO₄, filtered, and concentrated underreduced pressure. and the resulting residue was purified by silicachromatography (0 to 100% gradient of EtOAc/heptane) to give(1R,3R)-benzyl1-(((benzyloxy)carbonyl)amino)-3-hydroxy-8-azaspiro[4.5]decane-8-carboxylate(940 mg, 2.14 mmol) as a white foam. MS m/z 439.3 (M+H)⁺.

Step b: A mixture of (1R,3R)-benzyl1-(((benzyloxy)carbonyl)amino)-3-hydroxy-8-azaspiro[4.5]decane-8-carboxylate(440 mg, 1.003 mmol) and Dess-Martin periodinane (638 mg, 1.505 mmol) inDCM (6 mL) was stirred for 1 h at 0° C. and for 18 h at R T. Thereaction mixture was diluted with sat. aq NaHCO₃:sat. aq Na₂S₂O₃ (1:1,25 mL). The separated aqueous phase was extracted with DCM (3×15 mL),The combined organic phases were washed with brine, dried over MgSO₄,and concentrated under reduced pressure. The resulting residue waspurified by silica chromatography (0 to 70% gradient of EtOAc/heptane)to give (R)-benzyl1-(((benzyloxy)carbonyl)amino)-3-oxo-8-azaspiro[4.5]decane-8-carboxylate(415 mg, 0.951 mmol) as a white foam. MS m/z 437.2 (M+H)⁺.

Step c: To a solution of MeLi (1.2 M in THF, 2.61 mL, 3.13 mmol) in THF(15 mL) was added dropwise (R)-benzyl1-(((benzyloxy)carbonyl)amino)-3-oxo-8-azaspiro[4.5]decane-8-carboxylate(415 mg, 0.951 mmol) in THF (5 mL) at −30 to −40° C. The resultingmixture was stirred for 20 min at −30 to −40° C. The mixture was dilutewith NaHSO₄ (10% solution in H₂O), diluted with EtOAc, and allowed towarm up to RT under vigorously stirring. The mixture was diluted withsat. aq NaHCO₃ and The separated aqueous phase was extracted with EtOAc(1×15 mL). The combined organic phases were dried over Na₂SO₄, filtered,and concentrated under reduced pressure. A solution of the resultingresidue (313 mg), Na₂CO3 (498 mg, 4.70 mmol), and CbzCl (295 mg, 1.729mmol) in water (10 mL) and THF (1 mL) was vigorously stirred for 3 daysat RT. The mixture was diluted with EtOAc and the separated aqueousphase was extracted with EtOAc (3×15 mL). The combined organic phaseswere concentrated under reduced pressure. The resulting residue waspurified by silica chromatography (0 to 50% gradient of EtOAc/heptane)to give two diastereoners: diastereomer A (112 mg, 0.25 mmol) as acolorless semisolid, MS m/z 453.3 (M+H)⁺ and diastereomer B (45 mg,0.010 mmol) as white foam/solid, MS m/z 453.3 (M+H)⁺.

Step d: A mixture of diastereomer A (50 mg, 0.11 mmol) and Pd/C (10 wt.%; 12 mg, 0.011 mmol) in MeOH (8 mL) was stirred vigorously underhydrogen atmosphere for 2 h. Celite was added and the mixture wasfiltered through a pad of Celite followed by DCM wash. The filtrate wasconcentrated under reduced pressure to give(4R)-4-amino-2-methyl-8-azaspiro[4.5]decan-2-ol as a colorless solidwhich was used without further purification. MS m/z 185.2 (M+H)⁺.

Intermediate 40 (1R,3S)-tert-butyl1-((R)-1,1-dimethylethylsulfinamido)-3-fluoro-8-azaspiro[4.5]decane-8-carboxylate

A mixture (1R,3R)-tert-butyl1-((R)-1,1-dimethylethylsulfinamido)-3-hydroxy-8-azaspiro[4.5]decane-8-carboxylate(400 mg, 1.068 mmol) and DAST (1 M in DCM, 1.87 mL, 1.87 mmol) in DCM(8.5 mL) was stirred for 90 min at 0° C. The reaction mixture wasquenched by addition of sat. aq NaHCO₃ (5 mL). After stirring for 10 minat 0° C., the phases were separated and the aqueous was stracted withDCM (2×5 mL). The combined organic phases were dried over MgSO₄,filtered, and the volatiles were removed under reduced pressure to give(1R,3S)-tert-butyl1-((R)-1,1-dimethylethylsulfinamido)-3-fluoro-8-azaspiro[4.5]decane-8-carboxylatewhich was used in next step without further purification. MS m/z 277.2(M+H-Boc)⁺.

Intermediate 41 (1R,3R)-tert-butyl1-((R)-1,1-dimethylethylsulfinamido)-3-fluoro-8-azaspiro[4.5]decane-8-carboxylate

A mixture (1R,3S)-tert-butyl1-((R)-1,1-dimethylethylsulfinamido)-3-hydroxy-8-azaspiro[4.5]decane-8-carboxylate(200 mg, 0.534 mmol) and DAST (1 M in DCM, 934 μL, 0.934 mmol) in DCM (5mL) was stirred for 90 min at 0° C. The reaction mixture was quenched byaddition of sat. aq NaHCO₃ (5 mL). After stirring for 10 min at RT, thephases were separated and the aqueous was stracted with DCM (2×5 mL).The combined organic phases were dried over MgSO₄, filtered, and thevolatiles were removed under reduced pressure to give (1R,3R)-tert-butyl1-((R)-1,1-dimethylethylsulfinamido)-3-fluoro-8-azaspiro[4.5]decane-8-carboxylatewhich was used in next step without further purification. MS m/z 277.2(M+H-Boc)⁺.

Intermediate 423-((6-amino-2,3-dichloropyridin-4-yl)thio)-6-chloropyrazin-2-amine

Step a: To a solution of 5,6-dichloropyridin-2-amine (590 mg, 3.62 mmol)in THF (5 mL) was added LiHMDS (1 M in THF, 7.96 mL, 7.96 mmol) at 0° C.The reaction was stirred for 10 min at 0° C. then a solution of Boc₂O(869 mg, 3.98 mmol) in THF (5 mL) was added to reaction mixture. Theresulting solution was stirred for 15 min at 0° C. then taken to pH 4 byaddition of 1 M HCl. The solution was diluted with EtOAc, washed withsat. aq NaHCO₃, brine, dried over Na₂SO₄, filtered, and concentratedunder reduced pressure. The resulting residue was purified by silicachromatography (0 to 40% gradient of EtOAc/heptane) to give tert-butyl(5,6-dichloropyridin-2-yl)carbamate (790 mg, 3.00 mmol). ¹H NMR (400MHz, CHLOROFORM-d) δ ppm 7.86 (d, J=8.7 Hz, 1H), 7.70 (d, J=8.7 Hz, 1H),7.20 (br s, 1H), 1.51 (s, 9H). MS m/z 232.9 (M+H-tBu)⁺.

Step b: To a solution of diisopropylamine (1 mL, 7.07 mmol) in THF (5mL) was added n-BuLi (2.5 M in hexanes, 2.83 mL, 7.07 mmol) at −78° C.and the resulting solution was stirred for 1 h at this temperature.Tert-butyl (5,6-dichloropyridin-2-yl)carbamate (930 mg, 3.53 mmol) inTHF (5 mL) was added at −78° C. After stirring at this temperature for 2h, iodine (987 mg, 3.89 mmol) in THF (5 mL) was added and the resultingmixture was stirred for 30 min at −78° C. After warming to RT, thereaction mixture was diluted with water and extracted with EtOAc (2×50mL). The combined organic phases were washed with sat. aq Na₂S2O₃,brine, dried over Na₂SO₄, filtered, and concentrated under reducedpressure. The resulting residue was purified by silica chromatography (0to 40% gradient of EtOAc/heptane) to give tert-butyl(5,6-dichloro-4-iodopyridin-2-yl)carbamate (813 mg, 2.09 mmol). ¹H NMR(400 MHz, CHLOROFORM-d) δ ppm 8.45 (s, 1H), 7.12 (s, 1H), 1.52 (s, 9H).MS m/z 332.9 (M+H-tBu)⁺.

Step c: A mixture of tert-butyl(5,6-dichloro-4-iodopyridin-2-yl)carbamate (610 mg, 1.57 mmol), sodium3-amino-5-chloropyrazine-2-thiolate (302 mg, 1.65 mmol), Pd₂(dba)₃ (72mg, 0.08 mmol), Xantphos (91 mg, 0.16 mmol), and DIPEA (0.55 mL, 3.14mmol) in dioxane (7.8 mL) was stirred for 8 h at 110° C. After coolingto RT, the reaction mixture was filtered through a pad of Celite and thevolatiles were removed under reduced pressure. The resulting residue waspurified by silica chromatography (0 to 40% gradient of EtOAc/heptane)to give tert-butyl(4-((3-amino-5-chloropyrazin-2-yl)thio)-5,6-dichloropyridin-2-yl)carbamate(470 mg, 1.11 mmol). ¹H NMR (400 MHz, DMSO) δ ppm 10.24 (s, 1H), 7.96(s, 1H), 7.31 (br s, 2H), 7.16 (s, 1H), 1.38 (s, 9H). MS m/z 321.9(M+H-Boc)⁺.

Step d: A mixture of tert-butyl(4-((3-amino-5-chloropyrazin-2-yl)thio)-5,6-dichloropyridin-2-yl)carbamate(470 mg, 1.11 mmol) and HCl (4 M in dioxane, 5.56 mL, 22.24 mmol) wasstirred for 1 h at RT. The volatiles were removed under reduced pressureto give3-((6-amino-2,3-dichloropyridin-4-yl)thio)-6-chloropyrazin-2-aminedihydrochloride (411 mg, 1.04 mmol) which was used without furtherpurification. MS m/z 324.0 (M+H)⁺.

Example 1 (S) and (R)8-(6-amino-5-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrazin-2-yl)-8-azaspiro[4.5]decan-1-amine

Step a: A solution of6-chloro-3-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrazin-2-amine (200mg, 0.652 mmol) and N-(4-methoxybenzyl)-8-azaspiro[4.5]decan-1-amine(358 mg, 1.304 mmol) in DIPEA (3 mL) was stirred for 60 h at 130° C.After cooling to RT, the volatiles were removed under reduced pressure.The resulting residue was dissolved in TFA (3 mL) and the solution wasstirred in a microwave reactor for 1 h at 160° C. and for 15 min at 180°C. The volatiles were removed under reduced pressure and the resultingresidue was purified by HPLC (gradient elution 25-50% acetonitrile inwater, 5 mM NH₄OH modifier) to give8-(6-amino-5-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrazin-2-yl)-8-azaspiro[4.5]decan-1-amine(73 mg, 0.482 mmol; 83% pure based on HRMS). 19 mg of this compound werefurther purified by HPLC (gradient elution 25-50% acetonitrile in water,0.1% TFA modifier) to give the title compound pure (9.5 mg). ¹H NMR (400MHz, METHANOL-d₄) δ ppm 8.29 (dd, J=4.42, 1.39 Hz, 1H), 7.48 (s, 1H),7.19-7.41 (m, 2H), 4.06-4.26 (m, 2H), 2.89-3.14 (m, 2H), 2.71 (t, J=7.33Hz, 1H), 1.86-2.00 (m, 1H), 1.73-1.84 (m, 1H), 1.43-1.72 (m, 5H),1.27-1.42 (m, 2H), 1.17-1.27 (m, 1H). ¹⁹F NMR (376 MHz, METHANOL-d₄) δppm −66.45 (s). HRMS calcd for C₁₉H₂₄N₆F₃S (M+H)⁺425.1735, found425.1753. IC₅₀ is 0.023 μM.

Step b: Chiral SFC purification of the above title compound performed asfollows; column: ID 21×250 mm, flow rate: 75 g per minute, mobile phase:35% MeOH and 10 mM NH₄OH in CO₂, detection: 270 nm UV to obtain singleenantiomer R_(t) (P1)=4.9 min; IC₅₀ is 0.011 μM and R_(t) (P2)=6.4 min;IC₅₀ is 0.167 μM.

The following compounds of Formula I, as identified in table 4, weremade using the above procedure or modifications to the above procedureusing the corresponding tiopyrazin-2-amine derivative and protectedamine.

TABLE 4 Example Compound Characterization IC₅₀ (μM) 2

¹H NMR (400 MHz, DMSO-d₆) δ ppm 7.62 (s, 1 H), 7.39 (d, J = 7.83 Hz, 1H), 7.22 (t, J = 8.08 Hz, 1H), 6.57 (d, J = 7.83 Hz, 1 H), 6.13 (s, 2H), 4.03-4.21 (m, 2 H), 2.96- 3.13 (m, 2 H), 2.63-2.72 (m, 1 H),1.73-1.89 (m, 2 H), 1.48-1.69 (m, 3 H), 1.13-1.40 (m, 5 H). HRMS calcdfor C₁₉H₂₄Cl₂N₅S (M + H)⁺ 424.1129, found 424.1131. 0.025 3

¹H NMR (400 MHz, DMSO-d₆) δ ppm 7.63 (s, 1 H), 7.39 (d, J = 8.08 Hz, 1H), 7.22 (t, J = 7.96 Hz, 1H), 6.58 (d, J = 8.08 Hz, 1 H), 6.13 (s, 2H), 4.06-4.21 (m, 2 H), 2.96- 3.13 (m, 2 H), 2.71 (t, J = 7.45 Hz, 1 H),1.71-1.93 (m, 2 H), 1.45- 1.64 (m, 3 H), 1.13-1.42 (m, 5 H). HRMS calcdfor C₁₉H₂₄Cl₂N₅S (M + H)⁺ 424.1129, found 424.1129. 0.010 4

¹H NMR (400 MHz, DMSO-d₆) δ ppm 7.59 (s, 1 H), 7.39 (dd, J = 8.08, 1.26Hz, 1 H), 7.18-7.25 (m, 1 H), 6.57 (dd, J = 8.08, 1.52 Hz, 1 H), 6.11(s, 2 H), 3.94-4.05 (m, 2 H), 3.07-3.21 (m, 2 H), 2.40 (d, J = 4.55 Hz,1 H), 1.81-2.00 (m, 2 H), 1.13-1.67 (m, 9 H), 0.96-1.07 (m, 1 H). HRMScalcd for C₂₀H₂₆Cl₂N₅S (M + H)⁺ 438.1286, found 438.1283. 0.032 5

¹H NMR (400 MHz, CDCl₃) δ ppm 8.35 (dd, J = 4.5, 1.5 Hz, 1 H), 7.58 (s,1 H), 7.42 (d, J = 8.1 Hz, 1 H), 7.24-7.15 (m, 1 H), 4.77 (s, 2H), 4.13(m, 2 H), 3.90 (m, 1 H), 3.78 (m, 1 H), 3.28-3.17 (m, 2 H), 3.06 (t, J =6.9 Hz, 1 H), 2.27 (2, 1 H), 1.74-1.63 (m, 1 H), 1.63- 1.54 (m, 3 H),1.53-1.46 (m, 1 H), 1.21 (br. s, 2 H). HRMS calcd for C₁₈H₂₂F₃N₆OS (M +H)⁺ 427.1528, found 427.1526. 0.349

6

¹H NMR (400 MHz, DMSO-d₆) δ ppm 7.61 (dd, J = 7.96, 1.39 Hz, 1 H),7.43-7.48 (m, 1 H), 7.35-7.42 (m, 1 H), 7.27-7.33 (m, 1 H), 5.57 (br s.,2 H), 3.94 (dd, J = 12.76, 5.18 Hz, 2 H), 3.00-3.14 (m, 2 H), 2.40 (d, J= 4.55 Hz, 1 H), 1.79- 1.99 (m, 2 H), 1.15-1.66 (m, 9 H), 0.96-1.07 (m,1 H). HRMS calcd for C₂₀H₂₆Cl₂N₅ (M + H)⁺ 406.1565, found 406.1563 0.0747

¹H NMR (400 MHz, DMSO-d₆) δ ppm 7.61 (dd, J = 7.96, 1.64 Hz, 1 H), 7.48(s, 1 H), 7.35-7.42 (m, 1 H), 7.28-7.33 (m, 1 H), 5.53-5.63 (m, 2 H),4.01-4.13 (m, 2 H), 2.99 (qd, J = 12.38, 2.78 Hz, 2 H), 1.71- 1.91 (m, 2H), 1.47-1.66 (m, 4 H), 1.13-1.40 (m, 5 H). HRMS calcd for C₁₉H₂₄Cl₂N₅(M + H)⁺ 392.1409, found 392.1417. 0.073

Example 8 (R) and(S)-2-(6-amino-5-((2,3-dichlorophenyl)thio)pyrazin-2-yl)-2-azaspiro[3.3]heptan-5-amine

Step a: A solution of6-chloro-3-((2,3-dichlorophenyl)thio)pyrazin-2-amine (140 mg, 0.457mmol) and tert-butyl 2-azaspiro[3.3]heptan-5-ylcarbamate (HCl salt, 125mg, 0.502 mmol) in DIPEA (1 mL) was stirred for 24 h at 130° C. Aftercooling to RT, the volatiles were removed under reduced pressure. Theresulting residue was dissolved in DCM (5 mL), TFA (0.5 mL) was addedand the resulting mixture was stirred for 30 min at RT. The volatileswere removed under reduced pressure and the resulting residue waspurified by HPLC (gradient elution 25-50% acetonitrile in water, 5 mMNH₄OH modifier) to give2-(6-amino-5-((2,3-dichlorophenyl)thio)pyrazin-2-yl)-2-azaspiro[3.3]heptan-5-amine(75 mg, 0.186 mmol). ¹H NMR (400 MHz, METHANOL-d₄) δ ppm 7.31 (dd,J=8.03, 1.51 Hz, 1H), 7.18 (s, 1H), 7.11 (t, J=8.03 Hz, 1H), 6.60 (dd,J=8.03, 1.51 Hz, 1H), 4.45 (d, J=8.78 Hz, 1H), 4.03 (d, J=9.03 Hz, 1H),3.96 (d, J=9.03 Hz, 1H), 3.90 (d, J=8.78 Hz, 1H), 3.34-3.39 (partiallyoverlapped with solvent, m, 1H), 2.12-2.25 (m, 1H), 1.90-2.11 (m, 2H),1.52-1.67 (m, 1H). HRMS calcd for C₁₆H₁₈C₁₂N₅S (M+H)⁺382.0660, found382.0585. IC₅₀ is 5.36 μM.

Step b:2-(6-amino-5-((2,3-dichlorophenyl)thio)pyrazin-2-yl)-2-azaspiro[3.3]heptan-5-amine(53.9 mg, 0.141 mmol) was further purified by Chiral SFC; column: OJ-H21×250 mm, flow rate: 80 g per minute, mobile phase: 26% MeOH and 10 mMNH₄OH in CO₂, detection: 269 nm UV to obtain single enantiomer R_(t)(P1)=3.7 min; IC₅₀ is 17.49 μM and R_(t)(P2)=4.7 min.; IC₅₀ is 3.31 μM.

The following compounds of Formula I, as identified in table 5, weremade using the above procedure or modifications to the above procedureusing the corresponding pyrazin-2-amine derivative and protected amine.

TABLE 5 Example Compound Characterization IC₅₀ (μM) 9

¹H NMR (400 MHz, METHANOL-d₄) δ ppm 7.50 (dd, J = 7.91, 1.63 Hz, 1 H),7.29 (t, J = 7.91 Hz, 1 H), 7.21 (dd, J = 7.65, 1.63 Hz, 1 H), 6.98 (s,1 H), 4.29 (d, J = 8.53 Hz, 1 H), 3.89 (d, J = 8.53 Hz, 1 H), 3.82 (d, J= 8.53 Hz, 1 H), 3.76 (d, J = 8.78 Hz, 1 H), 3.27 (partially overlappedwith solvent, t, J = 8.41 Hz, 1 H), 2.03-2.16 (m, 1 H), 1.92-2.03 (m, 1H), 1.82-1.92 (m, 1 H), 1.45- 1.61 (m, 1 H). HRMS calcd for 1.106C₁₆H₁₈Cl₂N₅ (M + H)⁺ 350.0939, found 350.0876. 10a & 10b

 

Chiral SFC purification performed as follows; column: Cellulose LUX-2 21× 250 mm, flow rate: 75 g per minute, mobile phase: 45% MeOH and 10 mMNH₄OH in CO₂, detection: 354 nm UV to obtain single enantiomer R_(t)(P1) = 3.3 min, R_(t) (P2) = 5.6 min. ¹H NMR (400 MHz, METHANOL-d₄) δppm 7.61 (dd, J = 7.78, 1.76 Hz, 1 H), 7.40 (t, J = 7.78 Hz, 1 H), 7.35(dd, J = 7.53, 1.76 Hz, 1 H), 7.22 (s, 1 H), 3.80 (d, J = 10.79 Hz, 1H), 3.35-3.61 (m, 4 H), 2.18-2.36 (m, 1 H), 2.05-2.18 (m, 1 H), 1.95-2.05 (m, 1 H), 1.66-1.90 (m, 3 H). HRMS calcd for C₁₇H₂₀Cl₂N₅ (M + H)⁺364.1096, found 364.1078. P1 = 0.548 P2 = 0.189

11a & 11b

 

Chiral SFC purification performed as follows; column: AD-H 21 × 250 mm,flow rate: 75 g per minute, mobile phase: 50% MeOH and 10 mM NH₄OH inCO₂, detection: 354 nm UV to obtain single enantiomer R_(t) (P1) = 2.2min, R_(t) (P2) = 3.6 min. ¹H NMR (400 MHz, METHANOL-d₄) δ ppm 7.48 (dd,J = 7.78, 1.76 Hz, 1 H), 7.28 (t, J = 7.78 Hz, 1 H), 7.22 (dd, J = 7.53,1.76 Hz, 1 H), 7.04 (s, 1 H), 3.55 (br. s., 1 H), 3.28-3.42 (m, 4 H),2.05-2.20 (m, 2 H), 1.79-1.89 (m, 1 H), 1.60-1.73 (m, 3 H). HRMS calcdfor C₁₇H₂₀Cl₂N₅ (M + H)⁺ 364.1096, found 364.1082. P1 = 0.365 P2 = 0.145

12a & 12b

 

Chiral SFC purification performed as follows; column: AS-H 21 × 250 mm,flow rate: 75 g per minute, mobile phase: 40% IPA and 0.2% diethylaminein CO₂, detection: 354 nm UV to obtain single enantiomer R_(t) (P1) =2.0 min, R_(t) (P2) = 3.1 min. ¹H NMR (400 MHz, METHANOL-d₄) δ ppm 7.23(s, 1 H), 7.18 (dd, J = 8.03, 1.51 Hz, 1 H), 6.93-7.05 (m, 1 H), 6.50(dd, J = 8.03, 1.51 Hz, 1 H), 3.70 (d, J = 11.04 Hz, 1 H), 3.24-3.51 (m,3 H), 2.07-2.24 (m, 1 H), 1.94-2.07 (m, 1 H), 1.89 (d, J = 5.77 Hz, 1H), 1.54-1.82 (m, 4 H). HRMS calcd for C₁₇H₂₀Cl₂N₅S (M + H)⁺ 396.0816,found 396.0798. P1 = 5.787 P2 = 3.933

13a & 13b

 

Chiral SFC purification performed as follows; column: AS-H 21 × 250 mm,flow rate: 75 g per minute, mobile phase: 45% IPA and 0.2% diethylaminein CO₂, detection: 354 nm UV to obtain single enantiomer R_(t) (P1) =2.1 min, R_(t) (P2) = 3.5 mm. ¹H NMR (400 MHz, METHANOL-d₄) δ ppm 7.27-7.32 (m, 2 H), 7.11 (t, J = 7.91 Hz, 1 H), 6.60 (dd, J = 8.16, 1.38 Hz,1 H), 3.64-3.76 (m, 1 H), 3.39-3.57 (m, 4 H), 2.14-2.32 (m, 2 H),1.88-2.04 (m, 2 H), 1.68-1.88 (m, 2 H). HRMS calcd for C₁₇H₂₀Cl₂N₅S (M +H)⁺ 396.0816, found 396.0799. P1 = 0.354 P2 = 1.510

Example 147-(6-amino-5-((2,3-dichlorophenyl)thio)pyrazin-2-yl)-7-azaspiro[3.5]nonan-1-amine

A solution of 6-chloro-3-((2,3-dichlorophenyl)thio)pyrazin-2-amine (140mg, 0.457 mmol) and 2-(7-azaspiro[3.5]nonan-1-yl)isoindoline-1,3-dione(HCl salt, 154 mg, 0.502 mmol) in DIPEA (1 mL) was stirred for 16 h at130° C. After cooling to RT, the volatiles were removed under reducedpressure. A solution of the resulting residue and hydrazine hydrate (29μL, 0.602 mmol) in THF:MeOH (1:1, 1 mL) was stirred for 16 h at 55° C.After cooling to RT, the volatiles were removed under reduced pressureand the resulting residue was purified by HPLC (gradient elution 35-60%acetonitrile in water, 5 mM NH₄OH modifier) to give the title compound(78 mg, 0.502 mmol). ¹H NMR (400 MHz, METHANOL-d₄) δ ppm 7.59 (s, 1H),7.31 (dd, J=8.03, 1.51 Hz, 1H), 7.12 (t, J=8.03 Hz, 1H), 6.62 (dd,J=8.03, 1.51 Hz, 1H), 4.37 (d, J=13.55 Hz, 1H), 4.26 (d, J=13.55 Hz,1H), 3.24-3.30 (partially overlapped with solvent, m, 1H), 3.07-3.20 (m,1H), 2.92-3.06 (m, 1H), 2.26-2.39 (m, 1H), 1.87-2.07 (m, 2H), 1.57-1.87(m, 4H), 1.34-1.42 (m, 1H). HRMS calcd for C₁₈H₂₂C₁₂N₅S (M+H)⁺410.0973,found 410.1018; (racemic). IC₅₀ is 0.056 μM.

Chiral SFC purification of the above title compound performed asfollows; column: AD-H 21×250 mm, flow rate: 80 g per minute, mobilephase: 46% MeOH and 10 mM NH₄OH in CO₂, detection: 274 nm UV to obtainsingle enantiomer R_(t) (P1)=4.0 min and R_(t) (P2)=5.5 min. (P1(S-enantiomer (determined by X-ray)); IC₅₀ is 0.019 μM; (P2(R-enantiomer)); IC₅₀ is 0.414 μM.

The following compounds of Formula I, as identified in table 6, weremade using the above procedure or modifications to the above procedureusing the corresponding pyrazin-2-amine derivative andphthalamide-protected amine.

TABLE 6 Example Compound Characterization IC₅₀ (μM) 15

¹H NMR (400 MHz, METHANOL-d₄) δ ppm 7.53 (s, 1H), 6.86 (t, J = 8.0 Hz, 1H), 6.62 (dd, J = 1.3, 8.1 Hz, 1 H), 6.02 (dd, J = 1.3, 7.8 Hz, 1 H),4.37-4.10 (m, 2 H), 3.21-2.92 (m, 3 H), 2.32- 2.18 (m, 1 H), 1.92-1.62(m, 5 H), 1.61-1.49 (m, 2H). HRMS calcd for C₁₈H₂₄ClN₆S (M + H)⁺391.1472, found 391.1486. 0.016 16

¹H NMR (400 MHz, DMSO-d₆) δ ppm 7.61 (dd, J = 8.03, 1.51 Hz, 1 H), 7.49(s, 1 H), 7.38 (t, J = 7.78 Hz, 1 H), 7.30 (dd, J = 7.53, 1.51 Hz, 1 H),5.63 (s, 2 H), 4.03-4.19 (m, 1 H), 3.89-4.03 (m, 1 H), 3.34 (s, 2 H),3.05 (ddd, J = 13.55, 8.53, 5.52 Hz, 1 H), 2.86-2.99 (m, 2 H), 2.00-2.14(m, 1 H), 1.47-1.71 (m, 5 H), 1.28-1.47 (m, 2 H). HRMS calcd forC₁₈H₂₂Cl₂N₅ (M + H)⁺ 378.1252, found 378.1217. 0.145 17a & 17b

Chiral SFC purification performed as follows; column: AD-H 21 × 250 mm,flow rate: 75 g per minute, mobile phase: 45% IPA and 10 mM NH₄OH inCO₂, detection: 354 nm UV to obtain single enantiomer R_(t) (P1) = 3.4min, R_(t) (P2) = 4.6 min. ¹NMR (400 MHz, DMSO-d₆) δ ppm 7.61 (dd, J =8.03, 1.51 Hz, 1 H), 7.49 (s, 1 H), 7.38 (t, J = 7.78 Hz, 1 H), 7.30(dd, J = 7.53, 1.51 Hz, 1 H), 5.63 (s, 2 H), 4.03-4.19 (m, 1 H),3.89-4.03 (m, 1 H), 3.34 (s, 2 H), 3.05 (ddd, J = 13.55, 8.53, 5.52 Hz,1 H), 2.86-2.99 (m, 2 H), 2.00-2.14 (m, 1 H), 1.47-1.71 (m, 5 H),1.28-1.47 (m, 2 H). HRMS calcd for C₁₈H₂₂Cl₂N₅ (M + H)⁺ 378.1252, found378.1245. P1 = 0.120 P2 = 0.445 18

¹H NMR (400 MHz, METHANOL-d₄) δ ppm 7.53 (dd, J = 8.08, 1.77 Hz, 1 H)7.41 (s, 1 H) 7.31 (t, J = 7.83 Hz, 1 H) 7.23 (dd, J = 7.71, 1.64 Hz, 1H) 4.18 (m, 2 H), 3.73 (t, J = 7.20 Hz, 1 H) 3.44-3.61 (m, 2 H)3.26-3.44 (m, 2 H) 2.54 (dddd, J = 13.99, 9.32, 6.88, 4.42 Hz, 1 H)2.03- 2.29 (m, 3 H) 1.86-2.01 (m, 1 H) 1.68-1.86 (m, 1 H). HRMS calcdfor C₁₈H₂₂Cl₂N₅O₂S (M + H)⁺ 442.0871, found, 442.0848. 3.552 19

¹H NMR (400 MHz, METHANOL-d₄) δ ppm 7.52 (s, 1 H) 7.21 (dd, J = 8.08,1.52 Hz, 1 H) 7.01 (t, J = 7.96 Hz, 1 H) 6.55 (dd, J = 8.08, 1.26 Hz, 1H) 4.11- 4.29 (m, 2 H) 3.73 (t, J = 7.20 Hz, 1 H) 3.52 (dtd, J = 13.93,10.97, 10.97, 2.78 Hz, 2 H) 3.34-3.44 (m, 1 H) 3.25-3.34 (m, 1 H) 2.55(dddd, J = 13.93, 9.38, 6.88, 4.42 Hz, 1 H) 2.04-2.28 (m, 3 H) 1.85-2.00 (m, 1 H) 1.68-1.85 (m, 1 H). HRMS calcd for C₁₈H₂₂Cl₂N₅O₂S₂ [M +H)⁺ 474.0592, found 474.0569. 4.094

Example 20(S)-7-(6-amino-5-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrazin-2-yl)-7-azaspiro[3.5]nonan-1-amine

Step a: A mixture of6-chloro-3-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrazin-2-amine (230mg, 0.750 mmol) and(R)-2-methyl-N—((S)-7-azaspiro[3.5]nonan-1-yl)propane-2-sulfinamide (238mg, 0.975 mmol) in DIPEA (3.7 mL) was stirred for 10 h at 105° C. Aftercooling to RT, the volatiles were removed under reduced pressure and theresulting residue was purified by silica chromatography (5 to 70%gradient of EtOAc/heptane) to give(R)—N—((S)-7-(6-amino-5-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrazin-2-yl)-7-azaspiro[3.5]nonan-1-yl)-2-methylpropane-2-sulfinamide(172 mg, 0.334 mmol) as a white solid. MS m/z 515.2 (M+H)⁺.

Step b: A solution of(R)—N—((S)-7-(6-amino-5-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrazin-2-yl)-7-azaspiro[3.5]nonan-1-yl)-2-methylpropane-2-sulfinamide(142 mg, 0.376 mmol) and HCl (4 M in dioxane, 414 μL, 1.66 mmol) in DCM(1.4 mL) was stirred for 20 min at 40° C. After cooling to RT, HCl (1 Min H₂O) was added and the resulting aqueous mixture was extracted withDCM. The aqueous phase was basified with NH₄OH (28% in H₂O) until pH 12and it was extracted with DCM (3×20 mL). The combined organic layerswere washed with brine, dried over MgSO₄, filtered and the volatileswere removed under reduced pressure to give(S)-7-(6-amino-5-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrazin-2-yl)-7-azaspiro[3.5]nonan-1-amine(93 mg, 0.227 mmol). ¹H NMR (400 MHz, DMSO-d₆) δ ppm 8.40-8.53 (m, 1H),7.61-7.69 (m, 1H), 7.55 (dd, J=8.0, 4.5 Hz, 1H), 7.29 (d, J=8.0 Hz, 1H),6.19 (s, 2H), 4.11-4.24 (m, 1H), 3.99-4.06 (m, 1H), 3.06-3.20 (m, 2H),2.90-3.06 (m, 2H), 1.50-1.74 (m, 4H), 1.33-1.49 (m, 2H). HRMS calcd forC₁₈H₂₁F₃N₆S (M+H)⁺411.1566, found 411.1579. IC₅₀ is 0.038 μM.

Example 21(S)-5-amino-3-(1-amino-8-azaspiro[4.5]decan-8-yl)-6-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrazine-2-carboxamide

Step a: To a solution of6-chloro-3-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrazin-2-amine (1.2g, 2.119 mmol) in DCM (30 mL) was added at 0° C. NBS (745 mg, 4.19 mmol)in one portion. The resulting mixture was stirred vigorously for 30 minat 0° C. and for 1 h at RT. The clear solution was quenched with waterand extracted with DCM. The combined organic layers were subsequentlywashed with water, brine, dried over Na₂SO₄, filtered and concentrated.The resulting residue was purified by silica chromatography (0 to 50%gradient of EtOAc/heptane) to give5-bromo-6-chloro-3-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrazin-2-amine(938 mg, 2.51 mmol). MS m/z 387.2 (M+H)⁺.

Step b: A mixture of5-bromo-6-chloro-3-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrazin-2-amine(750 mg, 1.945 mmol) and copper(I) cyanide (348 mg, 3.89 mmol) in DMF (7mL) was stirred for 14 h at 120° C. After cooling to RT, the reactionwas filtered through a pad of Celite followed by MeOH (50 mL) wash. Thevolatiles were removed under reduced pressure and the resulting residuewas purified by silica chromatography (0 to 100% gradient ofEtOAc/heptane) to afford5-amino-3-chloro-6-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrazine-2-carbonitrile(301 mg, 0.907 mmol). MS m/z 332.3 (M+H)⁺.

Step c: A mixture of5-amino-3-chloro-6-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrazine-2-carbonitrile(52 mg, 0.157 mmol) and(S)—N—((R)-1-(4-methoxyphenyl)ethyl)-8-azaspiro[4.5]decan-1-amine (90mg, 0.314 mmol) in DIPEA (0.246 mL) was stirred for 1 h at 135° C. Aftercooling to RT, volatiles were removed under reduced pressure and theresulting residue was purified by silica chromatography (0 to 100%gradient of EtOAc/heptane) to afford5-amino-3-((S)-1-(((R)-1-(4-methoxyphenyl)ethyl)amino)-8-azaspiro[4.5]decan-8-yl)-6-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrazine-2-carbonitrile(77 mg, 0.132 mmol). MS m/z 584.5 (M+H)⁺.

Step d: A mixture of5-amino-3-((S)-1-(((R)-1-(4-methoxyphenyl)ethyl)amino)-8-azaspiro[4.5]decan-8-yl)-6-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrazine-2-carbonitrile(77 mg, 0.132 mmol) and NaOH (1 M in H₂O, 1.451 mL, 1.451 mmol) in MeOH(3.5 mL) was stirred in a microwave reactor for 35 min at 110° C. Aftercooling to RT, the volatiles were removed under reduced pressure to give5-amino-3-((S)-1-(((R)-1-(4-methoxyphenyl)ethyl)amino)-8-azaspiro[4.5]decan-8-yl)-6-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrazine-2-carboxamide(79 mg, 0.132 mmol) which was used in next step without furtherpurification. MS m/z 602.5 (M+H)⁺.

Step e: A solution of5-amino-3-((S)-1-(((R)-1-(4-methoxyphenyl)ethyl)amino)-8-azaspiro[4.5]decan-8-yl)-6-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrazine-2-carboxamide(79 mg, 0.132 mmol) in TFA (1.2 mL, 15.76 mmol) was stirred in amicrowave reactor at 100° C. until no starting material remained (3 h,monitored by LCMS). The volatiles were removed under reduced pressureand the resulting residue was purified by HPLC (gradient elution 25-50%acetonitrile in water, 5 mM NH₄OH modifier) to give(S)-5-amino-3-(1-amino-8-azaspiro[4.5]decan-8-yl)-6-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrazine-2-carboxamide(18.8 mg, 0.039 mmol). ¹H NMR (400 MHz, METHANOL-d₄) δ ppm 8.43 (dd,J=4.5, 1.4 Hz, 1H), 7.57 (dd, J=8.1, 1.3 Hz, 1H), 7.46 (dd, J=8.2, 4.5Hz, 1H), 3.92-3.88 (m, 2H), 3.20-3.08 (m, 2H), 2.77 (t, J=7.4 Hz, 1H),2.04-1.96 (m, 1H), 1.829-1.82 (m, 1H), 1.78-1.61 (m, 4H), 1.53 (ddd,J=12.3, 9.2, 5.7 Hz, 1H), 1.43 (ddd, J=9.8, 4.9, 2.0 Hz, 1H), 1.39-1.32(m, 1H), 1.30-1.23 (m, 1H). HRMS calcd for C₂₀H₂₅F₃N₇OS (M+H)⁺468.1715,found 468.1761; IC₅₀ is 0.010 μM.

The following compounds of Formula I, as identified in table 7, weremade using the above procedure or modifications to the above procedureusing the corresponding amine and amine deprotection method.

TABLE 7 Example Compound Characterization IC₅₀ (μM) 22

¹H NMR (400 MHz, METHANOL-d₄) δ ppm 8.33 (dd, J = 1.3, 4.5 Hz, 1 H),7.50- 7.43 (m, 1 H), 7.36 (dd, J = 4.5, 8.1 Hz, 1 H), 3.92-3.81 (m, 1H), 3.81-3.69 (m, 1 H), 3.11-3.00 (m, 1 H), 3.00-2.86 (m, 2 H), 2.18-2.06 (m, 1 H), 1.76-1.52 (m, 5 H), 1.48-1.36 (m, 2 H). HRMS calcd forC₁₉H₂₃F3N₇OS (M + H)⁺ 454.1673, found 454.1645 0.053

Example 23(S)-8-(5-amino-6-((2-(trifluoromethyl)pyridin-3-yl)thio)-1,2,4-triazin-3-yl)-8-azaspiro[4.5]decan-1-amine

Step a: A mixture of 3-chloro-1,2,4-triazin-5-amine (70 mg, 0.536 mmol),(S)—N—((R)-1-(4-methoxyphenyl)ethyl)-8-azaspiro[4.5]decan-1-amine (247mg, 0.644 mmol), and N-methylmorpholine (177 μL, 1.609 mmol) in MeCN (1mL) and NMP (0.1 mL) was irradiated in a microwave reactor for 45 min at90° C. After cooling to RT, the resulting residue was directly purifiedby silica chromatography (0 to 5% gradient of MeOH/DCM) to afford(S)-8-(5-amino-1,2,4-triazin-3-yl)-N—((R)-1-(4-methoxyphenyl)ethyl)-8-azaspiro[4.5]decan-1-aminethat was used in next step without further purification. MS m/z 383.5(M+H)⁺.

Step b: To a solution of(S)-8-(5-amino-1,2,4-triazin-3-yl)-N—((R)-1-(4-methoxyphenyl)ethyl)-8-azaspiro[4.5]decan-1-amine(194 mg, 0.507 mmol) in DCM (8 mL) was added, at 0° C., NBS (97 mg,0.543 mmol) in one portion. After stirring for 20 min at 0° C., theclear solution was quenched with few drops of Na₂CO₃ aq. and it wasextracted with DCM. The combined organic layer was dried over MgSO₄,filtered and the volatiles were removed under reduced pressure. Theresulting residue was purified by silica chromatography (0 to 100%gradient of EtOAc/heptane) to give(S)-8-(5-amino-6-bromo-1,2,4-triazin-3-yl)-N—((R)-1-(4-methoxyphenyl)ethyl)-8-azaspiro[4.5]decan-1-amine(77.9 mg, 0.169 mmol). MS m/z 463.4 (M+H)⁺.

Step c: A mixture of(S)-8-(5-amino-6-bromo-1,2,4-triazin-3-yl)-N—((R)-1-(4-methoxyphenyl)ethyl)-8-azaspiro[4.5]decan-1-amine(54.1 mg, 0.117 mmol), 2-(trifluoromethyl)pyridine-3-thiol (21 mg, 0.117mmol), XantPhos (7.46 mg, 0.013 mmol), Pd₂(dba)₃ (5.37 mg, 0.0058 mmol),and DIPEA (0.041 mL, 0.234 mmol) in dioxane (1 mL) was stirred in amicrowave reactor for 1.5 h at 130° C. After cooling to RT, the reactionwas filtered through a pad of Celite followed by EtOAc (10 mL) wash. Thevolatiles were removed under reduced pressure to give(S)-8-(5-amino-6-((2-(trifluoromethyl)pyridin-3-yl)thio)-1,2,4-triazin-3-yl)-N—((R)-1-(4-methoxyphenyl)ethyl)-8-azaspiro[4.5]decan-1-amine(65 mg, 0.116 mmol). MS m/z 560.5 (M+H)⁺.

Step d: A solution of(S)-8-(5-amino-6-((2-(trifluoromethyl)pyridin-3-yl)thio)-1,2,4-triazin-3-yl)-N—((R)-1-(4-methoxyphenyl)ethyl)-8-azaspiro[4.5]decan-1-amine(65 mg, 0.116 mmol) in TFA (1.253 mL, 16.26 mmol) was stirred at 100° C.until no starting material remained (1.5 h, monitored by LC/MS), thevolatiles were removed under reduced pressure, the resulting residue wasdiluted with water, and it was extracted with Et₂O (3×10 mL). Theaqueous layer was basified to pH 12 using NH₄OH (28% in water), and itwas extracted with DCM (3×10 mL). The combined organic layers werewashed with brine, dried over Na₂SO₄, filtered and the volatiles wereremoved under reduced pressure. The resulting residue was purified byHPLC (gradient elution 25-50% acetonitrile in water, 5 mM NH₄OHmodifier) to give(S)-8-(5-amino-6-((2-(trifluoromethyl)pyridin-3-yl)thio)-1,2,4-triazin-3-yl)-8-azaspiro[4.5]decan-1-amine(14.5 mg, 0.032 mmol). ¹H NMR (400 MHz, METHANOL-d₄) δ ppm 8.50-8.45 (m,1H), 7.60-7.54 (m, 1H), 7.53-7.46 (m, 1H), 4.64-4.50 (m, 2H), 3.22-3.09(m, 2H), 2.88 (t, J=7.3 Hz, 1H), 2.11-2.00 (m, 1H), 1.94-1.86 (m, 1H),1.84-1.74 (m, 1H), 1.74-1.63 (m, 3H), 1.59-1.46 (m, 2H), 1.45-1.39 (m,1H), 1.39-1.31 (m, 1H). HRMS calcd for C₁₈H₂₃F₃N₇S (M+H)⁺426.1688, found426.1667. IC₅₀ is 0.290 μM.

Example 24(S)-8-(4-amino-5-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrimidin-2-yl)-8-azaspiro[4.5]decan-1-amine

Step a: A mixture of 2-(trifluoromethyl)pyridine-3-thiol (150 mg, 0.837mmol), 2-chloro-5-iodopyrimidin-4-amine (267 mg, 1.047 mmol), XantPhos(53.3 mg, 0.092 mmol), Pd₂(dba)₃ (38.3 mg, 0.042 mmol), and DIPEA (0.292mL, 1.674 mmol) in dioxane (1 mL) was stirred in a microwave reactor for1.5 h at 130° C. After cooling to RT, the reaction was filtered througha pad of Celite followed by EtOAc (10 mL) wash. The volatiles wereremoved under reduced pressure to give2-chloro-5-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrimidin-4-amine (141mg, 0.460 mmol). MS m/z 307.4 (M+H)⁺.

Step b: A mixture of2-chloro-5-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrimidin-4-amine (70mg, 0.228 mmol) and(S)—N—((R)-1-(4-methoxyphenyl)ethyl)-8-azaspiro[4.5]decan-1-amine (105mg, 0.274 mmol) in DIPEA (0.359 mL) was stirred in a microwave reactorfor 1.5 h at 135° C. After cooling to RT, the volatiles were removedunder reduced pressure to give(S)-8-(4-amino-5-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrimidin-2-yl)-N—((R)-1-(4-methoxyphenyl)ethyl)-8-azaspiro[4.5]decan-1-amine(128 mg, 0.228 mmol). MS m/z 559.5 (M+H)⁺.

Step c: A solution of(S)-8-(4-amino-5-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrimidin-2-yl)-N—((R)-1-(4-methoxyphenyl)ethyl)-8-azaspiro[4.5]decan-1-amine(128 mg, 0.229 mmol) in TFA (2.471 mL, 32.1 mmol) was stirred at 100° C.until no starting material remained (1.5 h, monitored by LCMS), thevolatiles were removed under reduced pressure, the resulting residue wasdiluted with water, and it was then extracted with Et₂O (3×10 mL). Theaqueous layer was basified to pH 12 using NH₄OH (28% in water), and itwas extracted with DCM (3×10 mL). The combined organic layers werewashed with brine, dried over Na₂SO₄, filtered and the volatiles wereremoved under reduced pressure. The resulting residue was purified byHPLC (gradient elution 35-60% acetonitrile in water, 5 mM NH₄OHmodifier), to give(S)-8-(4-amino-5-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrimidin-2-yl)-8-azaspiro[4.5]decan-1-amine(32 mg, 0.072 mmol). ¹H NMR (400 MHz, METHANOL-d₄) δ ppm 8.41-8.35 (m,1H), 8.00 (s, 1H), 7.47-7.43 (m, 2H), 4.66-4.45 (m, 2H), 3.18-3.06 (m,2H), 2.81 (t, J=7.3 Hz, 1H), 2.09-1.97 (m, 1H), 1.94-1.86 (m, 1H),1.81-1.72 (m, 1H), 1.69-1.62 (m, 2H), 1.59-1.53 (m, 2H), 1.49-1.44 (m,1H), 1.40-1.35 (m, 1H), 1.33-1.25 (m, 1H). HRMS calcd for C₁₉H₂₄F₃N₆S(M+H)⁺425.1735, found 425.1741; IC₅₀ is 2.78 μM.

Example 25(R)-5-amino-3-(1-amino-8-azaspiro[4.5]decan-8-yl)-6-((3-chloro-2-(trifluoromethyl)pyridin-4-yl)thio)pyrazine-2-carboxamide

Step a: A solution of 5-amino-3-chloropyrazine-2-carbonitrile (1.55 g,10.0 mmol) and(R)—N—((R)-1-(4-methoxyphenyl)ethyl)-8-azaspiro[4.5]decan-1-amine (2.88g, 10.0 mmol) in DIPEA (10 μL) and NMP (5 mL) was stirred for 16 h at110° C. After cooling to RT, the reaction mixture was poured into aseparation funnel containing NaHCO₃ aq. and it was extracted with EtOAc.The combined organic phases were dried over MgSO₄, filtered and thevolatiles were removed under reduced pressure. The resulting residue waspurified by silica chromatography (0 to 5% gradient of MeOH/DCM) to give5-amino-3-((R)-1-(((R)-1-(4-methoxyphenyl)ethyl)amino)-8-azaspiro[4.5]decan-8-yl)pyrazine-2-carbonitrile(2.74 g, 6.74 mmol). MS m/z 407.3 (M+H)⁺.

Step b: (Note: This reaction was run in 4 batches of 500 mg each). Asolution of5-amino-3-((R)-1-(((R)-1-(4-methoxyphenyl)ethyl)amino)-8-azaspiro[4.5]decan-8-yl)pyrazine-2-carbonitrile(500 mg, 1.23 mmol) in MeOH (8 mL) and NaOH (2.5 M in H₂O, 5 mL, 12.3mmol) was stirred in a microwave reactor for 90 min at 130° C. Aftercooling to RT, the resulting mixture was purified by HPLC (35-60%gradient of acetonitrile/water, 5 mM NH₄OH modifier) to give5-amino-3-((R)-1-(((R)-1-(4-methoxyphenyl)ethyl)amino)-8-azaspiro[4.5]decan-8-yl)pyrazine-2-carboxamide(160 mg/reaction, 640 mg total, 1.51 mmol). MS m/z 425.3 (M+H)⁺.

Step c: A solution of5-amino-3-((R)-1-(((R)-1-(4-methoxyphenyl)ethyl)amino)-8-azaspiro[4.5]decan-8-yl)pyrazine-2-carboxamide(615 mg, 1.45 mmol) in TFA (11 mL) was stirred for 1 h at 100° C. Thevolatiles were removed under reduced pressure. A solution of theresulting residue, DIPEA (1.2 mL, 6.89 mmol), and Boc₂O (330 mg, 1.516mmol) in DCM (15 mL) was stirred for 2 h at RT. The volatiles wereremoved under reduced pressure and the resulting residue was purified bysilica chromatography (1 to 10% gradient of MeOH/DCM) to give(R)-tert-butyl(8-(6-amino-3-carbamoylpyrazin-2-yl)-8-azaspiro[4.5]decan-1-yl)carbamate(538 mg, 1.378 mmol). MS m/z 391.0 (M+H)⁺.

Step d: A solution of (R)-tert-butyl(8-(6-amino-3-carbamoylpyrazin-2-yl)-8-azaspiro[4.5]decan-1-yl)carbamate(538 mg, 1.378 mmol), and NBS (270 mg, 1.516 mmol) in DCM (5 mL) wasstirred for 20 min at 0° C. The reaction mixture was quenched with MeOH(2 mL) and stirred for 20 min at RT. The resulting mixture was pouredinto a separation funnel containing aq NaHCO₃. and it was extracted withDCM. The combined organic phases were dried over MgSO₄, filtered and thevolatiles were removed under reduced pressure to give (R)-tert-butyl(8-(6-amino-5-bromo-3-carbamoylpyrazin-2-yl)-8-azaspiro[4.5]decan-1-yl)carbamate(627 mg, 1.336 mmol). MS m/z 471.2 (M+H)⁺.

Step e: To a solution of (R)-tert-butyl(8-(6-amino-5-bromo-3-carbamoylpyrazin-2-yl)-8-azaspiro[4.5]decan-1-yl)carbamate(627 mg, 1.336 mmol), XantPhos (77 mg, 0.134 mmol), and Pd₂(dba)₃ (61.2mg, 0.067 mmol) in dioxane (3 mL) was added (at RT and under N₂)2-ethylhexyl-3-mercaptopropanoate (334 μL, 1.469 mmol) followed byaddition of DIPEA (467 μL, 2.67 mmol). The resulting solution wasstirred in a microwave reactor for 1 h at 90° C. After cooling to RT,the reaction was filtered through a pad of Celite followed by EtOAc (5mL) wash. The combined filtrates were concentrated and the resultingresidue was purified by silica chromatography (0 to 10% gradient ofMeOH/DCM) to give 2-ethylhexyl3-((3-amino-5-((R)-1-((tert-butoxycarbonyl)amino)-8-azaspiro[4.5]decan-8-yl)-6-carbamoylpyrazin-2-yl)thio)propanoate(574 mg, 0.946 mmol). MS m/z 607.4 (M+H)⁺.

Step f: To a solution of 2-ethylhexyl3-((3-amino-5-((R)-1-((tert-butoxycarbonyl)amino)-8-azaspiro[4.5]decan-8-yl)-6-carbamoylpyrazin-2-yl)thio)propanoate(574 mg, 0.946 mmol) in THF (3 mL) was added (at −78° C. and under N₂)potassium tert-butoxide (1 M in THF, 2.84 mL, 2.84 mmol). After stirringvigorously at −78° C. for 10 min, the reaction was quenched with K₂CO₃aq. (2 M, 500 μL) and the volatiles were removed under reduced pressure.The resulting residue was purified by HPLC (15 to 40% gradient ofacetonitrile/water, 5 mM NH₄OH modifier) to give (R)-tert-butyl(8-(6-amino-3-carbamoyl-5-mercaptopyrazin-2-yl)-8-azaspiro[4.5]decan-1-yl)carbamate(280 mg, 0.663 mmol). MS m/z 423.4 (M+H)⁺.

Step g: To a solution of (R)-tert-butyl(8-(6-amino-3-carbamoyl-5-mercaptopyrazin-2-yl)-8-azaspiro[4.5]decan-1-yl)carbamate(88 mg, 0.208 mmol), 3-chloro-4-iodo-2-(trifluoromethyl)pyridine (80 mg,0.260 mmol), XantPhos (12.1 mg, 0.021 mmol), and Pd₂(dba)₃ (9.6 mg, 0.01mmol) in dioxane (0.5 mL) was added (at RT and under N₂) DIPEA (110 L,0.625 mmol). The resulting solution was stirred in a microwave reactorfor 1 h at 90° C. After cooling to RT, the reaction was diluted withEtOAc and it was filtered through a pad of Celite followed by EtOAc (5mL) wash. The combined filtrates were concentrated and dried undervacuum. A solution of the resulting residue in DCM (1 mL) and TFA (400μL) was stirred for 10 min at RT. The volatiles were removed underreduce pressure and the resulting residue was purified by HPLC (gradientelution 25-50% acetonitrile in water, 5 mM NH₄OH modifier) to give(R)-5-amino-3-(1-amino-8-azaspiro[4.5]decan-8-yl)-6-((3-chloro-2-(trifluoromethyl)pyridin-4-yl)thio)pyrazine-2-carboxamide(60 mg, 0.120 mmol). ¹H NMR (400 MHz, METHANOL-d₄) δ ppm 8.18 (d, J=5.05Hz, 1H). 6.85 (d, J=5.31 Hz, 1H). 3.87 (t, J=13.89 Hz, 2H). 2.98-3.14(m, 2H), 2.72 (t, J=7.33 Hz, 1H). 1.86-2.02 (m, 1H). 1.73-1.81 (m, 1H).1.43-1.72 (m, 5H). 1.17-1.41 (m, 3H). ¹⁹F NMR (376 MHz, METHANOL-d₄) δppm −67.22 (s, 1F). HRMS calcd for C₂₀H₂₄ClF₃N₇OS (M+H)⁺502.1404, found502.1398. IC₅₀ is 0.058 μM.

Example 26(R)-8-(6-amino-5-((2-amino-3-chloropyridin-4-yl)thio)pyrazin-2-yl)-8-azaspiro[4.5]decan-1-amine

A mixture of3-((2-amino-3-chloropyridin-4-yl)thio)-6-chloropyrazin-2-amine (67 mg,0.233 mmol) and(R)-2-methyl-N—((R)-8-azaspiro[4.5]decan-1-yl)propane-2-sulfinamide (120mg, 0.465 mmol) in DIPEA (2 mL) was stirred for 5 h at 130° C. Aftercooling to RT, the volatiles were removed under reduced pressure. Asolution of the resulting residue in dioxane (5 mL) and HCl (4 M indioxane, 1 mL) was stirred for 1 h at 40° C. The volatiles were removeunder reduced pressure and the resulting residue was purified by HPLC(gradient elution 25-50% acetonitrile in water, 5 mM NH₄OH modifier).The resulting residue was further purified by SFC (Princeton DEAP 20×150mm, flow rate: 80 g per minute, mobile phase: 20-40% MeOH in CO₂ within5.7 min, mass triggered collection, oven temperature 40° C., backpressure 120 bar) to give(R)-8-(6-amino-5-((2-amino-3-chloropyridin-4-yl)thio)pyrazin-2-yl)-8-azaspiro[4.5]decan-1-amine(23 mg, 0.057 mmol). ¹H NMR (400 MHz, METHANOL-d₄) δ ppm 7.50-7.64 (m,2H), 5.91 (d, J=5.77 Hz, 1H), 4.26 (t, J=13.18 Hz, 2H), 3.03-3.20 (m,2H), 2.79 (t, J=7.53 Hz, 1H), 1.95-2.11 (m, 1H), 1.83-1.95 (m, 1H),1.52-1.82 (m, 5H), 1.37-1.52 (m, 2H), 1.32 (dd, J=13.30, 2.01 Hz, 1H).HRMS calcd for C₁₈H₂₅ClN₇S (M+H)⁺406.1581, found 406.1576. IC₅₀ is 0.014μM.

The following compounds were synthesized using the above procedure ormodifications to the above procedure using the corresponding amineprotected amine.

TABLE 8 Example Compound Characterization IC₅₀ (μM) 27

¹H NMR (400 MHz, METHANOL-d₄) δ ppm 7.50- 7.64 (m, 2 H), 5.90 (d, J =5.52 Hz, 1 H), 4.30 (d, J = 13.55 Hz, 1 H), 4.19 (d, J = 13.55 Hz, 1 H),2.94-3.19 (m, 3 H), 2.15-2.29 (m, 1 H), 1.60-1.89 (m, 5 H), 1.47-1.60(m, 2 H). HRMS calcd for C₁₇H₂₃ClN₇S (M + H)⁺ 392.1424, found, 392.1434.0.042

Example 28(R)-8-(6-amino-5-((3-chloropyridin-4-yl)thio)pyrazin-2-yl)-8-azaspiro[4.5]decan-1-amine

Step a: To a suspension of6-chloro-3-((3-chloropyridin-4-yl)thio)pyrazin-2-amine (53 mg, 0.194mmol) in DIPEA (2 mL) was added(R)-2-methyl-N—((R)-8-azaspiro[4.5]decan-1-yl)propane-2-sulfinamide (65mg, 0.252 mmol). The resulting mixture was stirred at 90° C. for 10 hand then concentrated. The crude was purified by silica chromatography(0-50% gradient of EtOAc/heptane; EtOAc containing 10% MeOH, heptanecontaining 2% Et₃N) to afford(R)—N—((R)-8-(6-amino-5-((3-chloropyridin-4-yl)thio)pyrazin-2-yl)-8-azaspiro[4.5]decan-1-yl)-2-methylpropane-2-sulfinamide(40 mg, 0.081 mmol) as an off-white solid. MS m/z 495.0 (M+H)⁺.

Step b: To a solution of(R)—N—((R)-8-(6-amino-5-((3-chloropyridin-4-yl)thio)pyrazin-2-yl)-8-azaspiro[4.5]decan-1-yl)-2-methylpropane-2-sulfinamide(40 mg, 0.081 mmol) in DCM (0.8 mL), a solution of HCl (4 M in dioxane,101 μL, 0.404 mmol) was added and the resulting mixture was stirred at40° C. for 1 h. An aquous solution of HCl (2 M, 2 mL) was added and theresulting mixture was extracted with DCM (2×). The aquous mixture wasbasified with ammonium hydroxide (28% in water) until pH=12 and it wasextracted with DCM (3×). The organic layers were combined, washed withbrine, dried with Na₂SO₄, filtered and concentrated to afford(R)-8-(6-amino-5-((3-chloropyridin-4-yl)thio)pyrazin-2-yl)-8-azaspiro[4.5]decan-1-amine(24 mg, 0.061 mmol). ¹H NMR (400 MHz, DMSO-d₆) δ ppm 8.49 (s, 1H), 8.25(d, J=5.3 Hz, 1H), 7.66 (s, 1H), 6.56 (d, J=5.3 Hz, 1H), 6.24 (s, 2H),4.07-4.26 (m, 2H), 2.98-3.13 (m, 2H), 2.70 (t, J=7.4 Hz, 1H), 1.11-1.94(m, 10H). HRMS calcd for C₁₈H₂₄ClN₆S (M+H)⁺391.1472, found 391.1480.IC₅₀ is 0.023 μM.

Example 29(R)-8-(6-amino-5-((2-chloropyridin-3-yl)thio)pyrazin-2-yl)-8-azaspiro[4.5]decan-1-amine

Step a: To a suspension of6-chloro-3-((2-chloropyridin-3-yl)thio)pyrazin-2-amine (85 mg, 0.311mmol) in DIPEA (1.6 mL), was added(R)-2-methyl-N—((R)-8-azaspiro[4.5]decan-1-yl)propane-2-sulfinamide (105mg, 0.405 mmol). The resulting mixture was stirred at 90° C. for 10 hand then concentrated. The crude was purified by silica chromatograpahy(0-50% gradient of EtOAc/heptane; EtOAc containing 10% MeOH, heptanecontaining 2% Et₃N) to afford(R)—N—((R)-8-(6-amino-5-((2-chloropyridin-3-yl)thio)pyrazin-2-yl)-8-azaspiro[4.5]decan-1-yl)-2-methylpropane-2-sulfinamide(40 mg, 0.081 mmol) as an off-white solid. ¹H NMR (400 MHz,CHLOROFORM-d) δ ppm 8.15 (dd, J=4.5, 1.8 Hz, 1H), 7.64 (s, 1H),7.01-7.18 (m, 2H), 4.87 (br. s, 2H), 4.24 (s, 2H), 3.29-3.45 (m, 1H),3.20 (d, J=5.8 Hz, 1H) 2.98-3.13 (m, 2H), 1.98-2.21 (m, 1H), 1.36-1.94(m, 9H), 1.22 (s, 9H). MS m/z 495.0 (M+H)⁺.

Step b: To a solution of(R)—N—((R)-8-(6-amino-5-((2-chloropyridin-3-yl)thio)pyrazin-2-yl)-8-azaspiro[4.5]decan-1-yl)-2-methylpropane-2-sulfinamide(66 mg, 0.133 mmol) in DCM (2 mL), was added a solution of HCl (4 M indioxane, 167 μL, 0.667 mmol) and the resulting mixture was stirred at40° C. for 1 h. An aqueous solution of HCl (2 M, 2 mL) was added and theresulting mixture was extracted with DCM (2×). The aqueous mixture wasbasified with ammonium hydroxide (28% in water) until pH=12 and it wasextracted with DCM (3×). The organic layers were combined, washed withbrine, dried with Na₂SO₄, filtered and concentrated to afford(R)-8-(6-amino-5-((2-chloropyridin-3-yl)thio)pyrazin-2-yl)-8-azaspiro[4.5]decan-1-amine(24 mg, 0.062 mmol) as a tan solid. ¹H NMR (400 MHz, METHANOL-d₄) δ ppm8.01 (dd, J=4.8, 1.8 Hz, 1H), 7.43-7.52 (m, 1H), 7.12 (dd, J=7.9, 4.6Hz, 1H), 7.00 (dd, J=7.9, 1.6 Hz, 1H), 4.11-4.26 (m, 2H), 2.96-3.10 (m,2H), 2.67-2.81 (m, 1H), 1.06-2.05 (m, 10H). HRMS calcd for C₁₈H₂₄ClN₆S(M+H)⁺391.1472, found 391.1470. IC₅₀ is 0.015 μM.

Example 30(R)-8-(6-amino-5-((2,3-dichloropyridin-4-yl)thio)pyrazin-2-yl)-8-azaspiro[4.5]decan-1-amine

Step a: To a suspension6-chloro-3-((2,3-dichloropyridin-4-yl)thio)pyrazin-2-amine (34 mg, 0.111mmol) in DIPEA (0.55 mL),(R)-2-methyl-N—((R)-8-azaspiro[4.5]decan-1-yl)propane-2-sulfinamide (37mg, 0.144 mmol) was added. The resulting mixture was stirred at 90° C.for 10 h and then concentrated. The crude was purified by silicachromatography (0-50% gradient of EtOAc/heptane; EtOAc containing 10%MeOH, heptane containing 2% Et₃N) to afford(R)—N—((R)-8-(6-amino-5-((2,3-dichloropyridin-4-yl)thio)pyrazin-2-yl)-8-azaspiro[4.5]decan-1-yl)-2-methylpropane-2-sulfinamide(33 mg, 0.062 mmol) as an off-white solid. ¹H NMR (400 MHz,CHLOROFORM-d) δ ppm 8.02 (d, J=5.3 Hz, 1H), 7.66 (s, 1H), 6.60 (d, J=5.3Hz, 1H), 4.82 (s, 2H), 4.21-4.34 (m, 2H), 3.34-3.42 (m, 1H), 3.20 (d,J=5.8 Hz, 1H), 2.99-3.15 (m, 2H), 2.08-2.21 (m, 1H), 1.26-1.97 (m, 9H),1.23 (s, 9H). MS m/z 529.1 (M+H)⁺.

Step b: To a solution of(R)—N—((R)-8-(6-amino-5-((2,3-dichloropyridin-4-yl)thio)pyrazin-2-yl)-8-azaspiro[4.5]decan-1-yl)-2-methylpropane-2-sulfinamide(20 mg, 0.038 mmol) in DCM (0.38 mL), was added a solution of HCl (4 Min dioxane, 47 μL, 0.189 mmol) and the resulting mixture was stirred at40° C. for 1 h. The reaction mixture was concentrated and dissolved inMeOH. The crude was purified by HPLC (gradient elution 15-40%acetonitrile in water, 5 mM NH₄OH modifier) to afford(R)-8-(6-amino-5-((2,3-dichloropyridin-4-yl)thio)pyrazin-2-yl)-8-azaspiro[4.5]decan-1-amine(7 mg, 0.016 mmol) as a white solid. ¹H NMR (400 MHz, METHANOL-d₄) δ ppm7.91-8.04 (m, 1H), 7.52-7.65 (m, 1H), 6.61 (d, J=5.3 Hz, 1H), 4.29 (t,J=14.2 Hz, 2H), 3.06-3.22 (m, 2H), 2.88 (t, J=7.4 Hz, 1H), 1.21-2.17 (m,10H). HRMS calcd for C₁₈H₂₃Cl₂N₆S (M+H)⁺425.1082, found 425.1095. IC₅₀is 0.003 μM.

Example 31(S)-7-(6-amino-5-((2,3-dichloropyridin-4-yl)thio)pyrazin-2-yl)-7-azaspiro[3.5]nonan-1-amine

Step a: To a suspension of6-chloro-3-((2,3-dichloropyridin-4-yl)thio)pyrazin-2-amine (54 mg, 0.176mmol) in DIPEA (1.8 mL), was added(R)-2-methyl-N—((R)-8-azaspiro[4.5]decan-1-yl)propane-2-sulfinamide (86mg, 0.351 mmol). The resulting mixture was stirred at 90° C. for 10 hand then concentrated. The crude was purified by silica chromatography(0-50% gradient EtOAc/heptane; EtOAc containing 10% MeOH, heptanecontaining 2% Et₃N) to afford(R)—N—((S)-7-(6-amino-5-((2,3-dichloropyridin-4-yl)thio)pyrazin-2-yl)-7-azaspiro[3.5]nonan-1-yl)-2-methylpropane-2-sulfinamide(52 mg, 0.102 mmol) as an off-white solid. MS m/z 514.9 (M+H)⁺.

Step b: To a solution of(R)—N—((S)-7-(6-amino-5-((2,3-dichloropyridin-4-yl)thio)pyrazin-2-yl)-7-azaspiro[3.5]nonan-1-yl)-2-methylpropane-2-sulfinamide(20 mg, 0.039 mmol) in DCM (0.38 mL), was added a solution of HCl (4 Min dioxane, 47 μL, 0.189 mmol) and the resulting mixture was stirred at40° C. for 1 h. The reaction mixture was concentrated and dissolved inMeOH. The crude was purified by HPLC (gradient elution 15-40%acetonitrile in water, 5 mM NH₄OH modifier) to afford(S)-7-(6-amino-5-((2,3-dichloropyridin-4-yl)thio)pyrazin-2-yl)-7-azaspiro[3.5]nonan-1-amine(7 mg, 0.017 mmol) as a white solid. ¹H NMR (400 MHz, METHANOL-d₄) δ ppm7.89 (d, J=5.5 Hz, 1H), 7.50 (s, 1H), 6.51 (d, J=5.5 Hz, 1H), 3.94-4.31(m, 2H), 2.76-3.11 (m, 3H), 2.06-2.24 (m, 1H), 1.36-1.82 (m, 7H). HRMScalcd for C₁₇H₂₁Cl₂N₆S (M+H)⁺411.0925, found 411.0938. IC₅₀ is 0.028 μM.

Example 32(R)-8-(6-amino-5-((2,3-dichloropyridin-4-yl)thio)pyrazin-2-yl)-8-azaspiro[4.5]decan-1-amine

Step a: To a suspension3-((3-amino-2-chlorophenyl)thio)-6-chloropyrazin-2-amine (60 mg, 0.209mmol) in DIPEA (1.5 mL) was added(R)-2-methyl-N—((R)-8-azaspiro[4.5]decan-1-yl)propane-2-sulfinamide (70mg, 0.272 mmol). The resulting mixture was stirred at 90° C. for 10 hand then concentrated. The crude was purified by silica chromatography(0-50% gradient of EtOAc/heptane; heptane containing 2% Et₃N) to afford(R)—N—((R)-8-(6-amino-5-((3-amino-2-chlorophenyl)thio)pyrazin-2-yl)-8-azaspiro[4.5]decan-1-yl)-2-methylpropane-2-sulfinamide(31 mg, 0.061 mmol) as an off-white solid. MS m/z 509.0 (M+H)⁺.

Step b: To a solution of(R)—N—((R)-8-(6-amino-5-((3-amino-2-chlorophenyl)thio)pyrazin-2-yl)-8-azaspiro[4.5]decan-1-yl)-2-methylpropane-2-sulfinamide(31 mg, 0.061 mmol) in DCM (0.6 mL) was added a solution of HCl (4 M indioxane, 76 μL, 0.304 mmol) and the resulting mixture was stirred at 40°C. for 1 h. An aqueous solution of HCl (2 M, 2 mL) was added and theresulting mixture was extracted with DCM (2×). The aqueous mixture wasbasified with ammonium hydroxide (28% in water) until pH=12 and it wasextracted with DCM (3×). The organic layers were combined, washed withbrine, dried over Na₂SO₄, filtered, and concentrated to afford(R)-8-(6-amino-5-((3-amino-2-chlorophenyl)thio)pyrazin-2-yl)-8-azaspiro[4.5]decan-1-amine(18 mg, 0.042 mmol) as a yellow solid. ¹H NMR (400 MHz, METHANOL-d₄) δppm 7.40 (s, 1H), 6.73 (t, J=8.0 Hz, 1H), 6.50 (dd, J=8.1, 1.3 Hz, 1H),5.90 (dd, J=7.8, 1.3 Hz, 1H), 4.02-4.18 (m, 2H), 3.21 (dt, J=3.2, 1.5Hz, 1H), 2.98 (d, J=11.4 Hz, 2H), 2.67 (t, J=7.5 Hz, 1H), 1.04-2.02 (m,10H). HRMS calcd for C₁₉H₂₆ClN₆S (M+H)⁺405.1628, found 405.1639. IC₅₀ is0.011 μM.

Example 33(R)-8-(6-amino-5-((3-chloro-2-fluoropyridin-4-yl)thio)pyrazin-2-yl)-8-azaspiro[4.5]decan-1-amine

To a suspension of6-chloro-3-((3-chloro-2-fluoropyridin-4-yl)thio)pyrazin-2-amine (40 mg,0.137 mmol) in DIPEA (0.7 mL) was added(R)-2-methyl-N—((R)-8-azaspiro[4.5]decan-1-yl)propane-2-sulfinamide (71mg, 0.0275 mmol). The resulting mixture was stirred at 90° C. for 10 hand then concentrated. The crude was dissolved in DCM (0.7 mL), asolution of HCl (4 M in dioxane, 34 μL, 0.137 mmol) was added and theresulting mixture was stirred at 40° C. for 1 h. The reaction mixturewas concentrated and the crude was purified by HPLC (gradient elution 15to 40% acetonitrile in water, 0.1% TFA modifier) to afford(R)-8-(6-amino-5-((3-chloro-2-fluoropyridin-4-yl)thio)pyrazin-2-yl)-8-azaspiro[4.5]decan-1-amine(TFA salt: 17 mg, 0.042 mmol). ¹H NMR (400 MHz, DMSO-d₆) δ ppm 7.94 (d,J=5.3 Hz, 1H), 7.79 (br. s., 3H), 7.69 (br. s., 1H), 6.51 (d, J=5.5 Hz,1H), 6.34 (br. s., 2H), 4.12-4.32 (m, 2H), 2.99-3.24 (m, 3H), 2.00-2.12(m, 1H), 1.30-1.90 (m, 9H). HRMS calcd for C₁₈H₂₃ClFN₆S (M+H)⁺409.1377,found 409.1385. IC₅₀ is 0.005 μM.

Example 34

Step a: To a RT solution of tert-butyl2-oxo-1,8-diazaspiro[4.5]decane-8-carboxylate (300 mg, 1.180 mmol) indichloromethane (3 mL) was added phosphorus pentasulfide (110 mg, 0.495mmol) followed by hexamethyldisiloxane (2.256 mL, 10.62 mmol). Thereaction was stirred for 3 h at RT then diluted with EtOAc and filteredthrough Celite. The filtrate was concentrated under reduced pressure.Crude product was purified by silica chromatography (0 to 80% gradientof EtOAc/heptane) giving tert-butyl1-thioxo-2,8-diazaspiro[4.5]decane-8-carboxylate (0.290 g, 1.07 mmol) asa white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 10.39 (s, 1H), 3.66 (dt,J=13.6, 4.9 Hz, 2H), 3.09 (s, 2H), 2.78 (t, J=7.8 Hz, 2H), 1.95 (t,J=7.8 Hz, 2H), 1.57 (dd, J=6.6, 4.8 Hz, 4H), 1.39 (s, 9H). MS m/z 271(M+H)⁺.

Step b: To a solution of1-thioxo-2,8-diazaspiro[4.5]decane-8-carboxylate (100 mg, 0.370 mmol) inTHF (3 mL) was added dropwise iodomethane (0.231 mL, 3.70 mmol). Theresulting solution was stirred at RT for 16 h. Throughout the course ofthe reaction the mixture slowly became more yellow in color and resultedin a light yellow precipitate after stirring the allotted reaction time.The reaction mixture was concentrated and dried under vacuum giving ayellow solid. The yellow solid was taken up in MeOH (2 mL) and treatedwith 7 M ammonia in methanol (3 mL) then heated in a sealed tube to 100°C. for 8 h. The reaction was cooled to RT and concentrated under reducedpressure giving a solid that was sonicated with acetonitrile andfiltered. The filtrate was concentrated and the residue purified bysilica chromatography (0 to 30% gradient of MeOH/DCM) giving tert-butyl1-amino-2,8-diazaspiro[4.5]dec-1-ene-8-carboxylate (87 mg, 0.343 mmol).¹H NMR (400 MHz, DMSO-d₆) δ 9.38 (s, 1H), 8.81 (d, J=25.2 Hz, 2H), 3.98(s, 2H), 3.55 (t, J=7.0 Hz, 2H), 2.82 (s, 2H), 2.12 (t, J=7.1 Hz, 2H),1.74 (td, J=12.9, 4.7 Hz, 2H), 1.57 (d, J=12.7 Hz, 2H), 1.41 (s, 9H). MSm/z 254 (M+H)⁺.

Step c: To a solution of tert-butyl1-amino-2,8-diazaspiro[4.5]dec-1-ene-8-carboxylate (86 mg, 0.339 mmol)in DCM (3 mL) was added HCl in dioxane (4 M, 0.500 mL, 2.0 mmol) at RTand the reaction stirred for 16 h. The reaction mixture was concentratedand residue was triturated from acetonitrile and filtered giving2,8-diazaspiro[4.5]dec-1-en-1-amine (57.7 mg, 0.254 mmol) as a tansolid. ¹H NMR (400 MHz, DMSO-d₆) δ 9.64 (s, 1H), 9.39-9.23 (m, 1H), 9.15(s, 1H), 9.07 (s, 1H), 8.70 (d, J=12.5 Hz, 1H), 3.54 (t, J=6.9 Hz, 2H),3.32 (d, J=13.3 Hz, 2H), 3.05-2.88 (m, 2H), 2.18 (t, J=6.9 Hz, 2H), 2.01(td, J=13.7, 4.3 Hz, 2H), 1.80 (d, J=13.8 Hz, 2H). MS m/z 154 (M+H)⁺.

Step d: To a suspension of6-chloro-3-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrazin-2-amine (250mg, 0.815 mmol) and 2,8-diazaspiro[4.5]dec-1-en-1-amine (210 mg, 1.371mmol) in N-methyl-2-pyrrolidinone (4 mL) was added DIPEA (1.4 mL, 8.02mmol) and the reaction heated to 140° C. for 16 h. The resulting darkmixture was cooled to RT and diluted with EtOAc and water. The layerswere partitioned and the organic discarded. The aqueous layer wasextracted with 20% isopropanol/chloroform mixture (2×30 mL), thecombined organics dried over Na₂SO₄, filtered and concentrated. Thecrude residue was purified using preparative HPLC (gradient elution, 5to 40% ACN in water, 0.1% TFA modifier) and half the pooled fractionswere lyophilized giving8-(6-amino-5-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrazin-2-yl)-2,8-diazaspiro[4.5]dec-1-en-1-amine(TFA salt: 61.4 mg, 0.082 mmol). The remaining fractions were combinedand neutralized by stirring for 10 minutes vigorously with 50% saturatedNaHCO₃. The resulting solution was extracted with 20%isopropanol/chloroform mixture (3×30 mL), the combined organics driedover Na₂SO₄, filtered and concentrated giving8-(6-amino-5-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrazin-2-yl)-2,8-diazaspiro[4.5]dec-1-en-1-amine(22 mg, 0.052 mmol) as the free base. ¹H NMR (400 MHz, DMSO-d₆) δ 8.46(d, J=4.4 Hz, 1H), 7.67 (s, 1H), 7.55 (dd, J=8.2, 4.5 Hz, 1H), 7.31 (d,J=8.1 Hz, 1H), 6.19 (s, 2H), 5.74 (s, 2H), 4.40 (d, J=13.4 Hz, 2H),3.43-3.34 (m, 2H), 2.90 (t, J=12.2 Hz, 2H), 1.97-1.89 (m, 2H), 1.83 (td,J=13.0, 4.1 Hz, 2H), 1.36 (d, J=12.9 Hz, 2H). MS m/z 424.1541 (M+H)⁺.IC₅₀ is 0.032 μM

The following compound was made using the above amine and coupled usingthe standard procedures described herein.

Example Compound Characterization IC₅₀ (μM) 35

¹H NMR (400 MHz, Chloroform-d) δ 8.35 (dd, J = 4.5, 1.5 Hz, 1 H), 7.58(s, 1 H), 7.42 (d, J = 8.1 Hz, 1 H), 7.24-7.15 (m, 1 H), 4.77 (s, 2 H),4.13 (m, 2 H), 3.90 (m, 1 H), 3.78 (m, 1 H), 3.28-3.17 (m, 2 H), 3.06(t, J = 6.9 Hz, 1 H), 2.27 (2, 1 H), 1.74-1.63 (m, 1 H), 1.63-1.54 (m, 3H), 1.53-1.46 (m, 1 H), 1.21 (br. s, 2 H). HRMS calcd for C₁₈H₂₂F₃N₆OS(M + H)⁺ 427.1528, found 427.1526. 0.232

Example 36racemic-8-(6-amino-5-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrazin-2-yl)-2-oxa-8-azaspiro[4.5]decan-4-amine

Dissolvedracemic-2-(8-(6-amino-5-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrazin-2-yl)-2-oxa-8-azaspiro[4.5]decan-4-yl)isoindoline-1,3-dione(40 mg, 0.072 mmol) in ethanol (1 mL) in a 5 mL conical microwave vial,added hydrazine hydrate (0.070 mL, 1.44 mmol), capped and heated on a90° C. aluminum bead bath for 2 h. Vacuum filtered suspension through0.45 μm PTFE membrane and washed with ethanol. HPLC purification(gradient elution 15 to 40% acetonitrile in water, 0.1% TFA modifier),then diluted with EtOAc and washed with sat. aq bicarbonate, then brine.Concentrated, diluted with 1 mL DCM and added HCl (100 μL, 4 M indioxane) to obtain precipitate. Concentrated to obtain8-(6-amino-5-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrazin-2-yl)-2-oxa-8-azaspiro[4.5]decan-4-amine (HClsalt: 1 mg, 0.002 mmol). ¹H NMR (400 MHz, Methanol-d₄) δ 8.43-8.39 (m,1H), 7.65 (s, 1H), 7.46-7.39 (m, 2H), 4.35-4.14 (m, 2H), 3.98 (d, J=9.2Hz, 1H), 3.90 (d, J=9.2 Hz, 1H), 3.58 (d, J=5.3 Hz, 1H), 3.29-3.12 (m,3H), 1.76 (m, 4H). HRMS calcd for C₁₈H₂₂F₃N₆OS (M+H)⁺427.1528, found427.1537. IC₅₀ is 0.07 μM.

Example 37 (R) and(S)-8-(6-amino-5-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrazin-2-yl)-2-oxa-8-azaspiro[4.5]decan-4-amine

Dissolved the single enantiomer P1,2-(8-(6-amino-5-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrazin-2-yl)-2-oxa-8-azaspiro[4.5]decan-4-yl)isoindoline-1,3-dione(49 mg, 0.088 mmol) in ethanol (1 mL) in a 5 mL conical microwave vial,added hydrazine hydrate (0.080 mL, 1.65 mmol), capped and heated on a90° C. aluminum bead bath for 2 h. Vacuum filtered the suspensionthrough 0.45 μm PTFE membrane and washed with ethanol. HPLC purification(gradient elution 15 to 40% acetonitrile in water, 5 mM NH₄OH modifier)resulted in the isolation of 8-(6-amino-5-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrazin-2-yl)-2-oxa-8-azaspiro[4.5]decan-4-amine (13mg, 0.029 mmol). Chiral analytical HPLC: LC-3 4.6×100 mm, 5 m, mobilephase: 45% MeOH with 10 mM ammonia, 5 mL/min, single enantiomer peak 1(P1), R_(t): 0.88 min, >99% single enantiomer. ¹H NMR (400 MHz,Methanol-d4) δ 8.39 (dd, J=4.3, 1.6 Hz, 1H), 7.60 (s, 1H), 7.41 (m, 2H),4.21-4.07 (m, 3H), 3.86 (d, J=8.7 Hz, 1H), 3.79 (d, J=8.7 Hz, 1H), 3.51(dd, J=9.0, 5.2 Hz, 1H), 3.24 (m, 2H), 3.15 (m, 1H), 1.73 (m, 2H), 1.59(m, 1.8 Hz, 2H). HRMS calcd for C₁₈H₂₂F₃N₆OS (M+H)⁺427.1528, found427.1542. IC₅₀ is 0.025 μM.

Example 38 (R) and(S)-8-(6-amino-5-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrazin-2-yl)-2-oxa-8-azaspiro[4.5]decan-4-amine

Dissolved the single enantiomer P2,2-(8-(6-amino-5-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrazin-2-yl)-2-oxa-8-azaspiro[4.5]decan-4-yl)isoindoline-1,3-dione(42 mg, 0.075 mmol) in ethanol (1 mL) in a 5 mL conical microwave vial,added hydrazine hydrate (0.080 mL, 1.65 mmol), capped and heated on a90° C. aluminum bead bath for 2 h. Vacuum filtered the suspensionthrough 0.45 μm PTFE membrane and washed with ethanol. HPLC purification(gradient elution 15 to 40% acetonitrile in water, 5 mM NH₄OH modifier)resulted in the isolation of 8-(6-amino-5-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrazin-2-yl)-2-oxa-8-azaspiro[4.5]decan-4-amine (13mg, 0.029 mmol). Chiral analytical HPLC: LC-3 4.6×100 mm, 5 m, mobilephase: 45% MeOH with 10 mM ammonia, 5 mL/min, single enantiomer peak 2(P2), R_(t): 1.33 min, >99% single enantiomer. ¹H NMR (400 MHz,Methanol-d4) δ 8.39 (dd, J=4.3, 1.6 Hz, 1H), 7.60 (s, 1H), 7.41 (m, 2H),4.21-4.07 (m, 3H), 3.86 (d, J=8.6 Hz, 1H), 3.79 (d, J=8.8 Hz, 1H), 3.50(dd, J=9.0, 5.2 Hz, 1H), 3.24 (m, 2H), 3.15 (m, 1H), 1.80-1.67 (m, 2H),1.64-1.50 (m, 2H). HRMS calcd for C₁₈H₂₂F₃N₆OS (M+H)⁺427.1528, found427.1536. IC₅₀ is 0.983 μM.

Example 39(R)-6-amino-2-(1-amino-8-azaspiro[4.5]decan-8-yl)-5-((2-(trifluoromethyl)pyridin-3-yl)thio)nicotinamide

Step a: To a solution of 2,6-dichloropyridine-3-carboxamide (0.728 g,3.81 mmol) in 1-methyl pyrrolidinone (7 mL) was added N-methylmorpholine (1.14 mL, 10.40 mmol) and(R)—N—((R)-1-(4-methoxyphenyl)ethyl)-8-azaspiro[4.5]decan-1-amine (1 g,3.47 mmol). The resulting mixture was heated to 100° C. under refluxingconditions for 24 h. The reaction mixture was diluted with ethylacetate, treated with concentrated sodium bicarbonate and filtered. Theorganic layer was separated, dried over sodium sulfate, filtered andconcentrated under reduced pressure. The resulting dark red oil waspurified by silica chromatography (0 to 50% gradient of ethylacetate/heptane containing 0.25% triethylamine) to furnish6-chloro-2-((R)-1-(((R)-1-(4-methoxyphenyl)ethyl)amino)-8-azaspiro[4.5]decan-8-yl)nicotinamide(0.998 g, 2.25 mmol). ¹H NMR (400 MHz, Methanol-d₄) δ 7.81 (d, J=7.8 Hz,1H), 7.26 (d, J=8.6 Hz, 2H), 6.86 (m, 3H), 3.82 (m, 1H), 3.77 (s, 3H),3.75-3.63 (m, 2H), 3.03 (m, 2H), 2.59 (m, 1H), 2.01-1.92 (m, 1H),1.88-1.52 (m, 5H), 1.51-1.36 (m, 3H), 1.32 (d, J=6.5 Hz, 3H), 1.25 (m,2H). MS m/z 442.9 (M+H)⁺.

Step b: To a solution of6-chloro-2-((R)-1-(((R)-1-(4-methoxyphenyl)ethyl)amino)-8-azaspiro[4.5]decan-8-yl)nicotinamide(242 mg, 0.546 mmol), in toluene (11 mL), was added Pd₂(dba)₃ (97 mg,0.169 mmol), and (oxybis(2,1-phenylene))bis(diphenylphosphine) (103 mg,0.191 mmol). The reaction mixture was sparged with nitrogen, andbenzophenone imine (0.11 mL, 0.656 mmol) and potassium tert-butoxide(0.710 mL, 1 M in tetrahydrofuran, 0.710 mmol) were added undernitrogen. The reaction mixture was heated to 80° C. for 2 h, and themixture was allowed to cool to RT, filtered through a pad of Celite, andwashed with ethyl acetate. The filtrate was concentrated under reducedpressure, and the residue was purified by silica chromatography (0 to50% gradient of ethyl acetate/heptane) to furnish6-((diphenylmethylene)amino)-2-((R)-1-(((R)-1-(4-methoxyphenyl)ethyl)amino)-8-azaspiro[4.5]decan-8-yl)nicotinamide(250 mg, 0.425 mmol). MS m/z 588.3 (M+H)⁺.

Step c: To a suspension of6-((diphenylmethylene)amino)-2-((R)-1-(((R)-1-(4-methoxyphenyl)ethyl)amino)-8-azaspiro[4.5]decan-8-yl)nicotinamide(130 mg, 0.221 mmol) in THF (6 mL), was added HCl (2 M, 0.1 mL, 0.200mmol) and the resulting solution stirred at RT for 30 minutes. Thereaction mixture was concentrated under reduced pressure and the residuewas purified by silica chromatography (0 to 50% gradient of ethylacetate/heptane, containing 0.25% triethylamine) to furnish6-amino-2-((R)-1-(((R)-1-(4-methoxyphenyl)ethyl)amino)-8-azaspiro[4.5]decan-8-yl)nicotinamide(43 mg, 0.102 mmol). MS m/z 424.1 (M+H)⁺.

Step d: A solution of6-amino-2-((R)-1-(((R)-1-(4-methoxyphenyl)ethyl)amino)-8-azaspiro[4.5]decan-8-yl)nicotinamide(199 mg, 0.470 mmol) in trifluoroacetic acid (3 mL) was heated to 100°C. for 30 min. The mixture was concentrated under reduced pressure andthe residue was used in the next step without further purification.(R)-6-amino-2-(1-amino-8-azaspiro[4.5]decan-8-yl)nicotinamide. MS m/z290.2 (M+H)⁺.

Step e: To a solution of(R)-6-amino-2-(1-amino-8-azaspiro[4.5]decan-8-yl)nicotinamide indichloromethane (2 mL) was added triethylamine (0.196 mL, 1.410 mmol)and di-tert-butyl dicarbonate (113 mg, 0.517 mmol) and the resultingmixture was stirred for 2 h at RT. The reaction mixture was concentratedunder reduced pressure, and the residue purified by silicachromatography (0 to 50% gradient of ethyl acetate/heptane, containing0.25% triethylamine) to furnish(R)-tert-butyl(8-(6-amino-3-carbamoylpyridin-2-yl)-8-azaspiro[4.5]decan-1-yl)carbamate(147 mg, 0.377 mmol). ¹H NMR (400 MHz, Methanol-d₄) δ 7.88 (d, J=8.5 Hz,1H), 6.19 (d, J=8.5 Hz, 1H), 3.66 (t, J=7.7 Hz, 1H), 3.27-3.15 (m, 2H),2.98 (t, J=12.4 Hz, 2H), 2.05-1.94 (m, 1H), 1.86-1.46 (m, 8H), 1.45 (s,9H), 1.41 (d, J=6.0 Hz, 1H). MS m/z 390.3 (M+H)⁺.

Step f: To a solution of (R)-tert-butyl(8-(6-amino-3-carbamoylpyridin-2-yl)-8-azaspiro[4.5]decan-1-yl)carbamate(136 mg, 0.349 mmol) in dichloromethane (5 mL), cooled on an ice bath,was added N-iodosuccinimide (86 mg, 0.384 mmol). The resulting mixturewas stirred at 5° C. for 2 h. The reaction was quenched by adding 2 mLof methanol, and the mixture allowed to warm up to RT. The solvents wereremoved under reduced pressure. The crude product was extracted withdichloromethane and washed with brine. The organic layer was dried oversodium sulfate, filtered and concentrated under reduced pressure toyield (R)-tert-butyl(8-(6-amino-3-carbamoyl-5-iodopyridin-2-yl)-8-azaspiro[4.5]decan-1-yl)carbamate(170 mg, 0.330 mmol) which was used in the next step without furtherpurification. MS m/z 516.1 (M+H)⁺.

Step g: To a solution of (R)-tert-butyl(8-(6-amino-3-carbamoyl-5-iodopyridin-2-yl)-8-azaspiro[4.5]decan-1-yl)carbamate(177 mg, 0.343 mmol) in dioxane (10 mL), was added Pd₂(dba)₃ (31.4 mg,0.034 mmol), (9,9-dimethyl-9H-xanthene-4,5-diyl)bis(diphenylphosphine)(39.7 mg, 0.069 mmol), 2-(trifluoromethyl)pyridine-3-thiol (67.7 mg,0.378 mmol), and N,N-diisopropylethylamine (0.120 mL, 0.687 mmol). Theresulting mixture was heated to 120° C. for 2 h, then allowed to cool toRT. The reaction mixture was diluted with ethyl acetate and filteredthrough a short pad of Celite. The filtrate was concentrated underreduced pressure and purified by silica chromatography (0 to 50%gradient of ethyl acetate/heptane, containing 0.25% triethylamine) tofurnish (R)-tert-butyl(8-(6-amino-3-carbamoyl-5-((2-(trifluoromethyl)pyridin-3-yl)thio)pyridin-2-yl)-8-azaspiro[4.5]decan-1-yl)carbamate(115 mg, 0.203 mmol). MS m/z 567.2 (M+H)⁺.

Step h: To a solution of (R)-tert-butyl(8-(6-amino-3-carbamoyl-5-((2-(trifluoromethyl)pyridin-3-yl)thio)pyridin-2-yl)-8-azaspiro[4.5]decan-1-yl)carbamate(110 mL, 0.194 mmol) in dichloromethane (2 mL) was added trifluoroaceticacid (2 mL, 26 mmol) and the resulting mixture was allowed to stir at RTfor 1 h. The solvents were removed under reduced pressure, and theresidue purified by HPLC (gradient elution: 35 to 60% acetonitrile inwater, 5 mM NH₄OH modifier) to furnish(R)-6-amino-2-(1-amino-8-azaspiro[4.5]decan-8-yl)-5-((2-(trifluoromethyl)pyridin-3-yl)thio)nicotinamide(40 mg, 0.084 mmol). ¹H NMR (400 MHz, Methanol-d₄) δ 8.38 (dd, J=4.1,1.9 Hz, 1H), 7.93 (s, 1H), 7.56-7.31 (m, 2H), 3.77-3.55 (m, 2H),3.16-2.98 (m, 2H), 2.82 (t, J=7.4 Hz, 1H), 2.03 (m, 1H), 1.94-1.60 (m,5H), 1.60-1.20 (m, 4 H). ¹⁹F NMR (376 MHz, Methanol-d₄) δ−66.48. HRMScalcd for C₂₁H₂₆F₃N₆OS (M+H)⁺467.1841, found 467.1837. IC₅₀ is 0.118 μM.

Example 40(R)-3-((3-amino-5-(1-amino-8-azaspiro[4.5]decan-8-yl)pyrazin-2-yl)thio)-2-chlorobenzamide

Step a: A mixture of 2-chloro-3-mercaptobenzamide (HCl salt, 145 mg,0.647 mmol), 3-bromo-6-chloropyrazin-2-amine (299 mg, 1.436 mmol),copper (I) iodide (49.3 mg, 0.259 mmol), potassium phosphate (412 mg,1.941 mmol), and 1,10-phenanthroline (58.3 mg, 0.324 mmol) in dioxane (5mL, degassed) was stirred in a microwave reactor for 4 h at 130° C.After cooling to RT, the reaction was filtered through a pad of Celitefollowed by EtOAc (50 mL) wash. The combined filtrates were concentratedand the resulting residue was purified by silica chromatography (0 to100% gradient of EtOAc/heptane) to afford3-((3-amino-5-chloropyrazin-2-yl)thio)-2-chlorobenzamide (140 mg, 0.444mmol). MS m/z 315.0 (M+H)⁺

Step b: A mixture of3-((3-amino-5-chloropyrazin-2-yl)thio)-2-chlorobenzamide (130 mg, 0.412mmol) and(R)-2-methyl-N—((R)-8-azaspiro[4.5]decan-1-yl)propane-2-sulfinamide (139mg, 0.536 mmol) in DIPEA (0.648 mL) was stirred in a microwave reactorfor 14 h at 95° C. After cooling to RT, the volatiles were removed underreduced pressure and the resulting residue was purified by silicachromatography (0 to 100% gradient of MeOH/DCM containing 0.25% TEA) toafford3-((3-amino-5-((R)-1-((R)-1,1-dimethylethylsulfinamido)-8-azaspiro[4.5]decan-8-yl)pyrazin-2-yl)thio)-2-chlorobenzamide(65 mg, 0.121 mmol). MS m/z 537.2 (M+H)⁺.

Step c:3-((3-amino-5-((R)-1-((R)-1,1-dimethylethylsulfinamido)-8-azaspiro[4.5]decan-8-yl)pyrazin-2-yl)thio)-2-chlorobenzamide(65 mg, 0.121 mmol) was dissolved in HCl/dioxane (4 M, 0.121 mL, 0.484mmol) and stirred at 22° C. until no starting material remained (1 h,monitored by LCMS). The volatiles were removed under reduced pressureand the resulting residue was purified by HPLC (gradient elution: 25 to50% acetonitrile in water, 5 mM NH₄OH modifier), to giveR)-3-((3-amino-5-(1-amino-8-azaspiro[4.5]decan-8-yl)pyrazin-2-yl)thio)-2-chlorobenzamide(25.5 mg, 0.058 mmol). ¹H NMR (400 MHz, DMSO-d₆) δ 7.90 (s, 1H), 7.63(s, 1H), 7.62 (br. s., 1H), 7.28-7.18 (m, 1H), 7.18-7.09 (m, 1H), 6.64(dd, J=1.6, 7.9 Hz, 1H), 6.08 (s, 2H), 4.18-4.07 (m, 2H), 3.12-2.95 (m,2H), 2.74-2.64 (m, 1H), 1.91-1.73 (m, 2H), 1.66-1.47 (m, 4H), 1.39-1.14(m, 4H). HRMS calcd for C₂₀H₂₆ClN₆OS (M+H)⁻ 433.1577, found 433.1598;IC₅₀ is 0.016 μM.

Example 41(2R,4R)-4-amino-8-(6-amino-5-((2-amino-3-chloropyridin-4-yl)thio)pyrazin-2-yl)-8-azaspiro[4.5]decan-2-ol

Step a: A mixture of (1R,3R)-tert-butyl3-((tert-butyldimethylsilyl)oxy)-1-((R)-1,1-dimethylethylsulfinamido)-8-azaspiro[4.5]decane-8-carboxylate(100 mg, 0.205 mmol) and HCl (4 M in dioxane, 510 μL, 2.05 mmol) in MeOH(1 mL) was stirred for 1 h at 40° C. The volatiles were removed underreduced pressure and the resulting white residue was dried under vaccumfor 1 h. MS m/z 171.1 (M+H)⁺.

Step b: A mixture of this white residue and3-((2-amino-3-chloropyridin-4-yl)thio)-6-chloropyrazin-2-amine (65 mg,0.226 mmol) in DIPEA:NMP (2:1; 1.5 mL) was vigorously stirred for 40 hat 100° C. After cooling to RT, the volatiles were removed under reducedpressure and the resulting crude was purified by HPLC (gradient elution7.5-20% acetonitrile in water, 0.1% TFA modifier). The volatiles wereremoved under reduced pressure and the resulting residue was furtherpurified by HPLC (gradient elution 15-40% acetonitrile in water, 5 mMNH₄OH modifier) to give(2R,4R)-4-amino-8-(6-amino-5-((2-amino-3-chloropyridin-4-yl)thio)pyrazin-2-yl)-8-azaspiro[4.5]decan-2-ol(44 mg, 0.102 mmol) as a white solid. ¹H NMR (400 MHz, METHANOL-d₄) δppm 7.51-7.64 (m, 2H), 5.92 (d, J=5.56 Hz, 1H), 4.16-4.39 (m, 3H),3.00-3.21 (m, 2H), 2.80 (dd, J=8.08, 7.07 Hz, 1H), 2.33 (dt, J=13.45,6.79 Hz, 1H), 1.95 (dd, J=13.89, 7.58 Hz, 1H), 1.83 (dd, J=14.02, 4.17Hz, 1H), 1.61-1.74 (m, 3H), 1.56 (ddd, J=13.39, 8.08, 5.81 Hz, 1H), 1.30(d, J=13.14 Hz, 1H). HRMS calcd for C₁₈H₂₅ClN₇OS (M+H)⁺422.1557, found422.1569. IC₅₀ is 0.007 μM.

The following compounds of table 9 were synthesized using the aboveprocedure or modifications to the above procedure using thecorresponding protected amine and chloro-pyrazine intermediate.

TABLE 9 Example Compound Characterization IC₅₀ (μM) 42

¹H NMR (400 MHz, METHANOL-d₄) δ ppm 8.39 (dd, J = 4.42, 1.39 Hz, 1 H),7.59 (s, 1 H), 7.22-7.50 (m, 2 H), 4.20-4.38 (m, 3 H), 2.97-3.22 (m, 2H), 2.84 (t, J = 7.45 Hz, 1 H), 2.34 (dt, J = 13.39, 6.69 Hz, 1 H),1.89-2.07 (m, 1 H), 1.78-1.89 (m, 1 H), 1.51-1.75 (m, 4 H), 1.32 (d, J =12.88 Hz, 1 H). ¹⁹F NMR (376 MHz, METHANOL- d₄) δ ppm −66.48. HRMS calcdfor C₁₉H₂₄F₃N₆OS (M + H)⁺ 441.1684, found 441.1657. 0.007 43

¹H NMR (400 MHz, METHANOL-d₄) δ ppm 8.34-8.47 (m, 1 H), 7.59 (s, 1 H),7.32-7.49 (m, 2 H), 4.34 (td, J = 6.69, 3.28 Hz, 3 H), 2.96-3.18 (m, 3H), 2.28 (dd, J = 14.15, 7.07 Hz, 1 H), 1.99 (ddd, J = 13.58, 6.88, 2.53Hz, 1 H), 1.68-1.87 (m, 2 H), 1.52-1.62 (m, 2 H), 1.49 (dd, J = 13.26,2.15 Hz, 1 H), 1.24-1.41 (m, 1 H). ¹⁹F NMR (376 MHz, METHANOL- d₄) δ ppm−66.48. HRMS calcd for C₁₉H₂₄F₃N₆OS (M + H)⁺ 441.1684, found 441.1651.0.006 44

¹H NMR (400 MHz, METHANOL-d₄) δ ppm 8.39 (dd, J = 4.42, 1.39 Hz, 1 H),7.59 (s, 1 H), 7.30-7.50 (m, 2 H), 4.15-4.42 (m, 3 H), 2.98-3.21 (m, 2H), 2.83 (t, J = 7.45 Hz, 1 H), 2.34 (dt, J = 13.45, 6.79 Hz, 1 H),1.89-2.04 (m, 1 H), 1.76-1.89 (m, 1 H), 1.51-1.76 (m, 4 H), 1.32 (d, J =12.88 Hz, 1 H). ¹⁹F NMR (376 MHz, METHANOL- d₄) δ ppm −66.48. HRMS calcdfor C₁₉H₂₄F₃N₆OS (M + H)⁺ 441.1684, found 441.1721. 0.135 45

¹H NMR (400 MHz, METHANOL-d₄) δ ppm 7.48-7.67 (m, 2 H), 5.92 (d, J =5.56 Hz, 1 H), 4.21-4.38 (m, 2 H), 3.92 (dt, J = 7.07, 3.54 Hz, 1 H),3.28 (s, 3 H), 2.94- 3.18 (m, 3 H), 2.25 (dd, J = 14.02, 7.20 Hz, 1 H),2.07 (ddd, J = 13.77, 6.95, 2.53 Hz, 1 H), 1.68-1.83 (m, 2 H), 1.51-1.68(m, 2 H), 1.42 (dd, J = 13.14, 2.27 Hz, 1 H), 1.28-1.38 (m, 1 H),1.08-1.20 (m, 2 H). HRMS calcd for C₁₉H₂₇ClN₇OS (M + H)⁺ 436.1686, found436.1666. 0.044 46

¹H NMR (400 MHz, METHANOL-d₄) δ ppm 7.99 (d, J = 5.31 Hz, 1 H), 7.60 (s,1 H), 6.61 (d, J = 5.31 Hz, 1 H), 4.24-4.42 (m, 2 H), 3.92 (tt, J =6.88, 3.35 Hz, 1 H), 3.28 (s, 3 H), 3.03-3.18 (m, 2 H), 3.00 (dd, J =8.72, 7.20 Hz, 1 H), 2.25 (dd, J = 14.15, 7.33 Hz, 1 H), 2.08 (ddd, J =13.83, 6.88, 2.53 Hz, 1 H), 1.68-1.82 (m, 2 H), 1.51- 1.68 (m, 2 H),1.44 (dd, J = 13.14, 2.27 Hz, 1 H), 1.29-1.40 (m, 1 H). HRMS calcd forC₁₉H₂₅Cl₂N₆OS (M + H)⁺ 455.1188, found 455.1166. 0.038 47

¹H NMR (400 MHz, METHANOL-d₄) δ ppm 8.39 (dd, J = 4.29, 1.26 Hz, 1 H),7.60 (s, 1 H), 7.30-7.51 (m, 2 H), 4.20-4.44 (m, 2 H), 2.94-3.20 (m, 3H), 2.35-2.61 (m, 2 H), 1.94-2.20 (m, 2 H), 1.81 (td, J = 12.69, 4.17Hz, 1 H), 1.69 (td, J = 12.69, 3.92 Hz, 1 H), 1.48-1.59 (m, 1 H), 1.44(dd, J = 13.39, 2.27 Hz, 1 H). ¹⁹F NMR (376 MHz, METHANOL-d₄) δ ppm−66.46, −84.76, −85.11. HRMS calcd for C₁₉H₂₂F₅N₆S (M + H)⁺ 461.1547,found 461.1548. 0.021 48

¹H NMR (400 MHz, METHANOL-d₄) δ ppm 8.38 (dd, J = 4.29, 1.26 Hz, 1 H),7.54- 7.63 (m, 1 H), 7.31-7.49 (m, 2 H), 4.15- 4.38 (m, 2 H), 3.81-3.96(m, 1 H), 3.27- 3.29 (m, 3 H), 3.02-3.19 (m, 2 H), 2.82 (t, J = 7.58 Hz,1 H), 2.34 (dt, J = 13.71, 6.92 Hz, 1 H), 1.93-2.06 (m, 1 H), 1.75-1.93(m, 1 H), 1.50-1.75 (m, 4 H), 1.24-1.39 (m, 1 H). ¹⁹F NMR (376 MHz,METHANOL-d₄) δ ppm −66.43. HRMS calcd for C₂₀H₂₆F₃N₆OS (M + H)⁺455.1841, found 455.1801. 0.009 49

¹H NMR (400 MHz, METHANOL-d₄) δ ppm 8.38 (dd, J = 4.29, 1.26 Hz, 1 H),7.58 (s, 1 H), 7.35-7.46 (m, 2 H), 4.21-4.40 (m, 3 H), 2.97-3.17 (m, 2H), 2.56 (dd, J = 9.22, 6.44 Hz, 1 H), 2.34-2.46 (m, 4 H), 1.91- 2.05(m, 1 H), 1.74-1.91 (m, 2 H), 1.68 (dd, J = 7.83, 3.79 Hz, 2 H), 1.56(ddd, J = 12.82, 9.16, 6.57 Hz, 1 H), 1.34 (d, J = 13.39 Hz, 1 H). ¹⁹FNMR (376 MHz, METHANOL-d₄) δ ppm −66.46. HRMS calcd for C₂₀H₂₆F₃N₆OS(M + H)⁺ 455.1841, found 455.1861. 0.234 50

¹H NMR (400 MHz, DMSO-d₆) δ 8.47 (dd, J = 4.4, 1.4 Hz, 1 H), 8.43 (s, 1H), 8.03 (s, 1 H), 7.70 (s, 1 H), 7.56 (dd, J = 8.2, 4.6 Hz, 1 H), 7.31(d, J = 8.1 Hz, 1 H), 6.23 (s, 2 H), 4.44 (d, J = 13.5 Hz, 2 H),2.97-2.80 (m, 2 H), 2.46 (s, 2 H), 1.94 (td, J = 13.1, 4.3 Hz, 2 H),1.48 (d, J = 12.6 Hz, 2H). HRMS calcd for C₁₈H₁₉F₃N₇OS (M + H)⁺438.1318, found 438.1418. 0.610 51

¹H NMR (400 MHz, METHANOL-d₄) δ ppm 7.76 (s, 1 H), 7.60 (s, 1 H), 5.90(s, 1 H), 4.27 (t, J = 13.01 Hz, 2 H), 3.08-3.21 (m, 2 H), 2.83 (t, J =7.45 Hz, 1 H), 1.99- 2.11 (m, 1 H), 1.29-1.96 (m, 9 H). HRMS calcd forC₁₈H₂₅ClN₇S (M + H)⁺ 406.1581, found 406.1595. 0.010 52

¹H NMR (400 MHz, METHANOL-d₄) δ 7.64-7.59 (m, 2 H), 6.14 (dd, J = 6.5,2.2 Hz, 1 H), 4.36-4.21 (m, 2 H), 4.18 (dd, J = 10.6, 5.6 Hz, 1 H), 3.97(d, J = 9.2 Hz, 1 H), 3.90 (d, J = 9.2 Hz, 1 H), 3.84 (dd, J = 10.7, 2.8Hz, 1 H), 3.58 (dd, J = 5.6, 2.8 Hz, 1 H), 3.28-3.11 (m, 2 H), 1.85-1.71(m, 4 H). HRMS calcd for C₁₇H₂₃ClN₇OS (M + H)⁺ 408.1373, found 408.1475.0.007 53

1H NMR (400 MHz, METHANOL-d₄) δ 7.65-7.57 (m, 2 H), 6.14 (dd, J = 6.5,2.2 Hz, 1 H), 4.34-4.21 (m, 2 H), 4.18 (dd, J = 10.6, 5.6 Hz, 1 H), 3.97(d, J = 9.2 Hz, 1 H), 3.90 (d, J = 9.2 Hz, 1 H), 3.84 (dd, J = 10.7, 2.8Hz, 1 H), 3.58 (dd, J = 5.6, 2.8 Hz, 1 H), 3.27-3.09 (m, 2 H), 1.85-1.71(m, 4 H). HRMS calcd for C₁₇H₂₃ClN₇OS (M + H)⁺ 408.1373, found 408.1474.0.298 54

¹H NMR (400 MHz, METHANOL-d₄) δ 8.10 (dd, J = 4.7, 1.8 Hz, 1 H), 7.58(s, 1 H), 7.21 (dd, J = 7.9, 4.7 Hz, 1 H), 7.11 (dd, J = 7.9, 1.7 Hz, 1H), 4.22-4.07 (m, 3 H), 3.86 (d, J = 8.6 Hz, 1 H), 3.79 (d, J = 8.7 Hz,1 H), 3.50 (dd, J = 9.0, 5.2 Hz, 1 H), 3.28- 3.11 (m, 3 H), 1.82-1.65(m, 2 H), 1.65- 1.50 (m, 2 H). HRMS calcd for C₁₇H₂₂ClN₆OS (M + H)⁺393.1264, found 393.1278. 0.010 55

¹H NMR (400 MHz, DMSO-d₆) δ 8.86 (d, J = 5.3 Hz, 1 H), 7.68 (s, 1 H),6.83 (d, J = 5.4 Hz, 1 H), 6.34 (s, 2 H), 4.11-3.90 (m, 3 H), 3.65 (dd,J = 46.4, 8.4 Hz, 2 H), 3.27-3.16 (m, 3 H), 3.05 (t, J = 6.2 Hz, 1 H),1.73-1.49 (m, 3 H), 1.47-1.35 (m, 2 H). HRMS calcd for C₁₆H₂₁ClN₇OS (M +H)⁺ 394.1217, found 394.0713. 0.073 56

 

Major diastereomer: ¹H NMR (400 MHz, METHANOL-d₄) δ 7.68-7.48 (m, 2 H),5.92 (d, J = 5.5 Hz, 1 H), 4.21-4.04 (m, 2 H), 3.28-3.10 (m, 2 H), 2.79(d, J = 5.9 Hz, 1 H), 2.44-2.23 (m, 1 H), 1.94-1.40 (m, 8 H), 1.01 (d, J= 7.0 Hz, 3 H). HRMS calcd for C₁₉H₂₇ClN₇S (M + H)⁺ 420.1737, found420.1748. Minor diastereomer: ¹H NMR (400 MHz, METHANOL-d₄) δ 7.66-7.49(m, 2 H), 5.92 (d, J = 5.5 Hz, 1 H), 4.36 (t, J = 12.0 Hz, 2 H),3.16-2.90 (m, 2 H), 2.17 (d, J = 10.0 Hz, 1 H), 1.94-1.53 (m, 6 H),1.37- 1.27 (m, 3 H), 1.08 (d, J = 6.5 Hz, 3 H). HRMS calcd forC₁₉H₂₇ClN₇S (M + H)⁺ 420.1737, found 420.1748. 0.009 57

¹H NMR (400 MHz, METHANOL-d₄) δ ppm 7.99 (d, J = 5.56 Hz, 1 H), 7.61 (s,1 H), 6.61 (d, J = 5.31 Hz, 1 H), 4.36 (dt, J = 6.63, 3.38 Hz, 3 H),2.99-3.19 (m, 3 H), 2.28 (dd, J = 14.02, 6.95 Hz, 1 H), 1.95- 2.08 (m, 1H), 1.72-1.90 (m, 2 H), 1.44- 1.68 (m, 3 H), 1.36 (d, J = 11.12 Hz, 1H). HRMS calcd for C₁₈H₂₃Cl₂N₆OS (M + H)⁺ 441.1031, found 441.0937.0.003 58

¹H NMR (400 MHz, METHANOL-d₄) δ ppm 7.99 (d, J = 5.31 Hz, 1 H), 7.61 (s,1 H), 6.61 (d, J = 5.31 Hz, 1 H), 4.23-4.39 (m, 3 H), 3.01-3.22 (m, 2H), 2.85 (t, J = 7.33 Hz, 1 H), 2.34 (dt, J = 13.39, 6.69 Hz, 1 H), 1.97(dd, J = 14.15, 7.33 Hz, 1 H), 1.85 (dd, J = 14.02, 3.92 Hz, 1 H),1.50-1.74 (m, 4 H), 1.33 (d, J = 13.64 Hz, 1 H). HRMS calcd forC₁₈H₂₃Cl₂N₆OS 441.1031 (M + H)⁺, found 441.1020. 0.004 59

¹H NMR (400 MHz, METHANOL-d₄) δ ppm 7.51-7.64 (m, 2 H) 5.91 (d, J = 5.56Hz, 1 H) 4.19-4.37 (m, 2 H) 3.83-3.96 (m, 1 H) 3.28 (s, 3 H) 3.01-3.20(m, 2 H) 2.80 (t, J = 7.58 Hz, 1 H) 2.34 (dt, J = 13.71, 6.92 Hz, 1 H)1.94-2.04 (m, 1 H) 1.78-1.90 (m, 1 H) 1.53-1.75 (m, 4 H) 1.30 (d, J =13.39 Hz, 1 H). HRMS calcd for C₁₉H₂₆ClN₆OS (M + H)⁺ 436.1686, found436.1663. 0.010 60

¹H NMR (400 MHz, METHANOL-d₄) δ ppm 7.51-7.64 (m, 2 H) 5.91 (d, J = 5.56Hz, 1 H) 4.19-4.37 (m, 2 H) 3.83-3.96 (m, 1 H) 3.28 (s, 3 H) 3.01-3.20(m, 2 H) 2.80 (t, J = 7.58 Hz, 1 H) 2.34 (dt, J = 13.71, 6.92 Hz, 1 H)1.94-2.04 (m, 1 H) 1.78-1.90 (m, 1 H) 1.53-1.75 (m, 4 H) 1.30 (d, J =13.39 Hz, 1 H). HRMS calcd for C₁₉H₂₆ClN₆OS (M + H)⁺ 436.1188, found455.1234. 0.004 61

Chiral SFC purification performed as follow; column: ADH 21 × 250 mm,flow rate: 80 g per minute, mobile phase: 30% IPA and 5 mM NH₄OH in CO₂,detection: 260 nm UV to obtain two peaks R_(t) (Peak 1) = 3.5 min. NMRas a mixture of diastereomers (as depicted). Major diastereomer: ¹H NMR(400 MHz, METHANOL-d₄) δ ppm 8.38 (dd, J = 4.42, 1.39 Hz, 1 H),7.51-7.66 (m, 1 H), 7.33- 7.48 (m, 2 H), 4.14-4.38 (m, 2 H), 2.95- 3.21(m, 2 H), 2.72-2.95 (m, 1 H), 2.19- 2.34 (m, 1 H), 1.86-2.19 (m, 1 H),1.52- 1.81 (m, 3 H), 1.24-1.48 (m, 3 H), 0.93- 1.20 (m, 4 H). HRMS calcdfor C₂₀H₂₆F₃N₆S (M + H)⁺ 439.1892, found 439.1897. 0.007 62

Chiral SFC purification performed as follow; column ADH 21 × 250 mm,flow rate: 80 g per minute, mobile phase: 30% IPA and 5 mM NH₄OH in CO₂,detection: 260 nm UV to obtain two peaks R_(t) (Peak 2) = 4.5 min. NMRwas a mixture of diastereomers (as depicted). Major diastereomer: ¹H NMR(400 MHz, METHANOL-d₄) δ ppm 8.38 (dd, J = 4.55, 1.26 Hz, 1 H),7.54-7.66 (m, 1 H), 7.32- 7.49 (m, 2 H), 4.15-4.39 (m, 2 H), 2.98- 3.19(m, 2 H), 2.76-2.98 (m, 1 H), 2.19- 2.31 (m, 1 H), 1.91-2.14 (m, 1 H),1.52- 1.81 (m, 3 H), 1.26-1.49 (m, 3 H), 0.99- 1.21 (m, 4 H). HRMS calcdfor C₂₀H₂₆F₃N₆S (M + H)⁺ 439.1892, found 439.1927. 0.100 63

¹H NMR (400 MHz, DMSO-d₆) δ ppm 8.10 (d, J = 5.3 Hz, 1 H), 7.67 (s, 1H), 6.52-6.62 (m, 1 H), 6.32 (br. s, 2 H), 3.97- 4.11 (m, 2 H), 3.95(dd, J = 8.5, 6.5 Hz, 1 H), 3.71 (d, J = 8.5 Hz, 1 H), 3.59 (d, J = 8.3Hz, 1 H), 3.29 (dd, J = 8.5, 5.8 Hz, 1 H), 3.15-3.25 (m, 2 H), 3.04 (t,J = 6.1 Hz, 1 H), 1.51-1.70 (m, 4 H), 1.35-1.46 (m, 2 H). HRMS calcd forC₁₇H₂₁Cl₂N₆OS (M + H)⁺ 427.3513, found 427.0852. 0.007 64

¹H NMR (400 MHz, DMSO-d₆) δ ppm 7.93 (d, J = 5.0 Hz, 1 H), 7.67 (s, 1H), 6.52 (d, J = 5.5 Hz, 1 H), 6.32 (s, 2 H), 3.97-4.10 (m, 2 H), 3.95(dd, J = 8.5, 6.8 Hz, 1 H), 3.71 (d, J = 8.5 Hz, 1 H), 3.60 (d, J = 8.3Hz, 1 H), 3.30 (dd, J = 8.5, 5.8 Hz, 1 H), 3.20 (ddd, J = 13.4, 10.5,3.0 Hz, 2 H), 3.05 (t, J = 6.1 Hz, 1 H), 1.52-1.70 (m, 2 H), 1.41 (td, J= 8.8, 4.6 Hz, 2 H). HRMS calcd for C₁₇H₂₁ClFN₆OS (M + H)⁺ 411.1170,found 411.1165. 0.013 65

¹H NMR (400 MHz, METHANOL-d₄) δ ppm 8.09 (s, 1 H), 7.62 (s, 1 H), 6.21(d, J = 1.8 Hz, 1 H), 4.30 (t, J = 13.5 Hz, 2 H), 3.22-3.07 (m, 2 H),2.82 (br s, 1 H), 2.09- 1.98 (m, 1 H), 1.95-1.86 (m, 1 H), 1.83- 1.55(m, 5 H), 1.52-1.28 (m, 3 H). HRMS calcd for C₁₈H₂₃ClFN₆S (M + H)⁺409.1377, found 409.1369. 0.310 66

¹H NMR (400 MHz, METHANOL-d₄) δ ppm 7.99 (d, J = 5.52 Hz, 1 H), 7.61 (s,1 H), 6.61 (d, J = 5.27 Hz, 1 H), 5.19 (td, J = 6.84, 3.39 Hz, 1 H),4.20-4.44 (m, 2 H), 2.96-3.19 (m, 3 H), 2.52 (quin, J = 6.96 Hz, 1 H),2.38 (dd, J = 14.56, 7.28 Hz, 1 H), 1.98-2.11 (m, 1 H), 1.86-1.98 (m, 1H), 1.72-1.86 (m, 1 H), 1.54-1.72 (m, 2 H), 1.33-1.51 (m, 2 H),1.05-1.18 (m, 6 H). HRMS calcd for C₂₂H₂₉Cl₂N₆O₂S (M + H)⁺ 511.1450,found 511.1453. 0.065 67

¹H NMR (400 MHz, METHANOL-d₄) δ ppm 7.53-7.64 (m, 2 H), 5.91 (d, J =5.56 Hz, 1 H), 4.24-4.43 (m, 2 H), 2.95-3.18 (m, 3 H), 2.36-2.61 (m, 2H), 1.95-2.19 (m, 2 H), 1.81 (td, J = 12.76, 4.29 Hz, 1 H), 1.69 (td, J= 12.69, 3.92 Hz, 1 H), 1.54 (dd, J = 13.26, 2.15 Hz, 1 H), 1.43 (dd, J= 13.39, 2.53 Hz, 1 H). ¹⁹F NMR (376 MHz, METHANOL-d₄) δ ppm −84.69,−85.07. HRMS calcd for C₁₈H₂₃ClF₂N₇O₂S (M + H)⁺ 442.1392, found442.1443. 0.010

Example 68(3R,4S)-8-(6-amino-5-((2-amino-3-chloropyridin-4-yl)thio)pyrazin-2-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decan-4-amine

Step a: A mixture of (3R,4S)-tert-butyl4-((R)-1,1-dimethylethylsulfinamido)-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-carboxylate(53 mg, 0.142 mmol) and HCl (4 M in dioxane, 354 μL, 1.415 mmol) in MeOH(5 mL) was stirred for 1 h at 40° C. After cooling to RT, the volatileswere removed under reduced pressure to give(3R,4S)-3-methyl-2-oxa-8-azaspiro[4.5]decane-4-amine which was used innext step without further purification. MS m/z 171.1 (M+H)⁺.

Step b: A mixture of(3R,4S)-3-methyl-2-oxa-8-azaspiro[4.5]decane-4-amine crude,3-((2-amino-3-chloropyridin-4-yl)thio)-6-chloropyrazin-2-amine (35.5 mg,0.123 mmol), and DIPEA (193 μL, 1.11 mmol) in DMSO (600 μL) was stirredfor 16 h at 100° C. After cooling to RT, the volatiles were removedunder reduced pressure and the resulting residue was purified by HPLC(gradient elution 15-40% acetonitrile in water, 5 mM NH₄OH modifier) togive(3R,4S)-8-(6-amino-5-((2-amino-3-chloropyridin-4-yl)thio)pyrazin-2-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decan-4-amine(13 mg, 0.030 mmol). ¹H NMR (400 MHz, METHANOL-d₄) δ ppm 7.72-7.51 (m,2H), 5.92 (d, J=5.5 Hz, 1H), 4.31 (m, 2H), 4.01-3.78 (m, 2H), 3.58 (dq,J=8.1, 6.0 Hz, 1H), 3.04 (m, 2H), 2.48 (d, J=8.1 Hz, 1H), 1.75 (m, 2H),1.61-1.47 (m, 2H), 1.31 (d, J=6.1 Hz, 3H). HRMS calcd for C₁₈H₂₅ClN₇OS(M+H)⁺422.1530, found 422.1505. IC₅₀ is 0.010 μM.

Example 69(3S,4S)-8-(6-amino-5-((2-amino-3-chloropyridin-4-yl)thio)pyrazin-2-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decan-4-amine

Step a: A mixture of (3S,4S)-tert-butyl4-((R)-1,1-dimethylethylsulfinamido)-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-carboxylate(51 mg, 0.136 mmol) and HCl (4 M in dioxane, 340 μL, 1.362 mmol) in MeOH(5 mL) was stirred for 1 h at 40° C. After cooling to RT, the volatileswere removed under reduced pressure to give(3S,4S)-3-methyl-2-oxa-8-azaspiro[4.5]decane-4-amine which was used innext step without further purification. MS m/z 171.1 (M+H)⁺.

Step b: A mixture of(3S,4S)-3-methyl-2-oxa-8-azaspiro[4.5]decane-4-amine crude,3-((2-amino-3-chloropyridin-4-yl)thio)-6-chloropyrazin-2-amine (35.5 mg,0.123 mmol), and DIPEA (193 μL, 1.11 mmol) in DMSO (600 μL) was stirredfor 16 h at 100° C. After cooling to RT, the volatiles were removedunder reduced pressure and the resulting residue was purified by HPLC(gradient elution 15-40% acetonitrile in water, 5 mM NH₄OH modifier) togive(3S,4S)-8-(6-amino-5-((2-amino-3-chloropyridin-4-yl)thio)pyrazin-2-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decan-4-amine(11 mg, 0.026 mmol). ¹H NMR (400 MHz, METHANOL-d₄) δ ppm 7.67-7.47 (m,2H), 5.91 (d, J=5.5 Hz, 1H), 4.22 (qd, J=6.4, 4.8 Hz, 1H), 4.03 (ddt,J=13.5, 8.9, 4.7 Hz, 2H), 3.86 (d, J=8.7 Hz, 1H), 3.71 (d, J=8.7 Hz,1H), 3.37 (td, J=9.9, 4.9 Hz, 1H), 3.29-3.23 (m, 1H), 3.00 (d, J=5.0 Hz,1H) 1.91-1.56 (m, 4H), 1.21 (d, J=6.4 Hz, 3H). HRMS calcd forC₁₈H₂₅ClN₇OS (M+H)⁺422.1530, found 422.1514. IC₅₀ is 0.010 μM.

Example 70(1R,3R)-8-(6-amino-5-((2-amino-3-chloropyridin-4-yl)thio)pyrazin-2-yl)-3-methyl-8-azaspiro[4.5]decan-1-amine

Step a: A mixture of (1R,3R)-benzyl1-((R)-1,1-dimethylethylsulfinamido)-3-methyl-8-azaspiro[4.5]decane-8-carboxylate(100 mg, 0.246 mmol) and HCl (4 M in dioxane, 1.5 mL, 6.5 mmol) in MeOH(1.5 mL) was stirred in a microwave reactor for 14 h at 140° C. Aftercooling to RT, the volatiles were removed under reduced pressure to give(1R,3R)-3-methyl-8-azaspiro[4.5]decan-1-amine which was used in nextstep without further purification. MS m/z 169.2 (M+H)⁺.

Step b: A mixture of (1R,3R)-3-methyl-8-azaspiro[4.5]decan-1-amine crude(theor 0.246 mmol) and3-((2-amino-3-chloropyridin-4-yl)thio)-6-chloropyrazin-2-amine (70.9 mg,0.246 mmol) in DIPEA (1 mL) and DMSO (0.5 mL) was stirred for 2 h at130° C. After cooling to RT, the volatiles were removed under reducedpressure and the resulting residue was purified by HPLC (gradientelution 15-40% acetonitrile in water, 5 mM NH₄OH modifier) to give(1R,3R)-8-(6-amino-5-((2-amino-3-chloropyridin-4-yl)thio)pyrazin-2-yl)-3-methyl-8-azaspiro[4.5]decan-1-amine(23 mg, 0.055 mmol). ¹H NMR (400 MHz, METHANOL-d₄) δ ppm 7.51-7.64 (m,2H), 5.91 (d, J=5.31 Hz, 1H), 4.18-4.37 (m, 2H), 3.02-3.18 (m, 2H), 2.82(dd, J=9.60, 6.32 Hz, 1H), 2.09-2.20 (m, 1H), 2.00-2.09 (m, 1H),1.91-2.00 (m, 1H), 1.58-1.74 (m, 2H), 1.24-1.48 (m, 3H), 1.09-1.20 (m,1H), 1.01-1.09 (m, 3H). HRMS cald for C₁₉H₂₇ClN₇S (M+H)⁺420.1737, found420.1719. IC₅₀ is 0.005 μM.

Example 71(1R,3S)-8-(6-amino-5-((2-amino-3-chloropyridin-4-yl)thio)pyrazin-2-yl)-3-methyl-8-azaspiro[4.5]decan-1-amine

Step a: A suspension of (1R,3S)-benzyl1-((R)-1,1-dimethylethylsulfinamido)-3-methyl-8-azaspiro[4.5]decane-8-carboxylate(600 mg, 1.476 mmol) and Pd(OH)₂ (104 mg, 0.148 mmol) in EtOAc:THF (1:275 mL) was stirred vigorously under H₂ atmosphere for 48 h. The reactionmixture was filtered through a pad of Celite followed by MeOH (50 mL)wash. The volatiles were removed under reduced pressure. A solution ofthe resulting residue and HCl (4 M in dioxane, 1.0 mL, 4.0 mmol) wasstirred for 2 h at 45° C. After cooling to RT, the volatiles wereremoved under reduced pressure. A suspension of the resulting residueand Pd/C (10% in charcoal, 200 mg) in MeOH (20 mL) was shaked for 2 hunder 60 psi H₂ atmosphere. The reaction mixture was filtered through apad of Celite followed by MeOH (50 mL) wash. The volatiles were removedunder reduced pressure to afford(1R,3S)-3-methyl-8-azaspiro[4.5]decan-1-amine which was used in nextstep without further purification. MS m/z 169.1 (M+H)⁺.

Step b: A mixture of (1R,3S)-3-methyl-8-azaspiro[4.5]decan-1-amine crude(0.729 mmol) and3-((2-amino-3-chloropyridin-4-yl)thio)-6-chloropyrazin-2-amine (150 mg,0.521 mmol) in DIPEA (3.2 mL) and DMA (6 mL) was stirred for 14 h at100° C. After cooling to RT, the volatiles were removed under reducedpressure and the resulting residue was purified by HPLC (gradientelution 10-30% acetonitrile in water, 0.1% TFA modifier) to give crudesolid. This crude solid was further purified by HPLC (gradient elution15-40% acetonitrile in water, 5 mM NH₄OH modifier) to give(1R,3S)-8-(6-amino-5-((2-amino-3-chloropyridin-4-yl)thio)pyrazin-2-yl)-3-methyl-8-azaspiro[4.5]decan-1-amine(80 mg, 0.189 mmol). ¹H NMR (400 MHz, METHANOL-d₄) δ ppm 7.65-7.49 (m,2H), 5.91 (d, J=5.5 Hz, 1H), 4.30 (ddt, J=12.4, 9.7, 3.6 Hz, 2H), 3.34(s, 1H), 3.19-2.95 (m, 1H), 2.92-2.80 (m, 1H), 2.34-2.16 (m, 2H),1.85-1.49 (m, 4H), 1.41 (dq, J=13.5, 2.7 Hz, 1H), 1.30 (dq, J=13.5, 2.6Hz, 1H), 1.13-0.92 (m, 4H). HRMS cald for C₁₉H₂₇ClN₇S (M+H)⁺420.1737,found 420.1716. IC₅₀ is 0.005 μM.

Example 728-(6-amino-5-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrazin-2-yl)-1-oxa-3,8-diazaspiro[4.5]dec-2-en-2-amine

Step a: A solution of6-chloro-3-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrazin-2-amine (70mg, 0.304 mmol), tert-butyl ((4-hydroxypiperidine-4-yl)methyl)carbamate(103 mg, 0.336 mmol), and DIPEA (2.0 mL, 11.45 mmol) in NMP (1 mL) wasstirred for 3 h at 120° C. After cooling to RT, the reaction was dilutedwith EtOAc, the organic phase was washed with water, brine, dried overNa₂SO₄, and the volatiles were removed under reduced pressure to give abrown oily residue. This residue was taken up in DCM (5 mL) and HCl (4 Min dioxane; 760 μL, 3.04 mmol) was added in two portions (half at thebeginning of the reaction and the other half 3 h later). The reactionwas stirred a total of 4 h. The volatiles were removed under reducedpressure and the resulting residue was triturated with MeCN to give abrown solid. The resulting crude was freebased suspending in 5% MeOH/DCMand adding sat. aq NaHCO₃. The resulting layers were separated and theaqueous was extracted again with 5% MeOH/DCM. The combine organic phaseswere concentrated under reduced pressure to give1-(6-amino-5-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrazin-2-yl)-4-(aminomethyl)piperidin-4-ol(65 mg, 0.149 mmol) as an off-white-tan solid. ¹H NMR (400 MHz, DMSO-d₆)δ ppm 8.47 (dd, J=4.6, 1.4 Hz, 1H), 7.68 (s, 1H), 7.55 (dd, J=8.3, 4.5Hz, 1H) 7.32 (dd, J=8.3, 1.4 Hz, 1H), 4.04 (dt, J=13.8, 4.2 Hz, 2H),3.38-3.28 (m, 2H), 2.83 (s, 2H), 1.70-1.48 (m, 4H). MS m/z 401.2 (M+H)⁺.

Step b: A solution of1-(6-amino-5-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrazin-2-yl)-4-(aminomethyl)piperidin-4-ol(65 mg, 0.162 mmol) in EtOH (3 mL) was treated in succession withcyanogen bromide (0.541 mL, 1.623 mmol) followed by NaHCO₃ (68.2 mg,0.812 mmol) and the resulting mixture was stirred for 16 h at RT. Thevolatiles were removed under reduced pressure and the resulting residuewas purified by HPLC (gradient elution 15-40% acetonitrile in water, 5mM NH₄OH modifier) to give8-(6-amino-5-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrazin-2-yl)-1-oxa-3,8-diazaspiro[4.5]dec-2-en-2-amine(12.5 mg, 0.029 mmol). ¹H NMR (400 MHz, DMSO-d₆) δ ppm 8.46 (dd, J=4.5,1.4 Hz, 1H), 7.68 (s, 1H), 7.55 (dd, J=8.3, 4.6 Hz, 1H), 7.31 (dd,J=8.2, 1.5 Hz, 1H), 6.22 (s, 2H), 5.78 (s, 2H), 3.94-3.73 (m, 2H),3.64-3.45 (m, 2H), 3.36 (s, 2H), 1.88-1.56 (m, 4H). HRMS calcd forC₁₇H₁₉F3N₇OS (M+H)⁺426.1318, found 426.1296. IC₅₀ is 0.193 μM.

The following compounds of table 10 were synthesized using the aboveprocedure or modifications to the above procedure using thecorresponding protected amine and chloro-pyrazine intermediate.

TABLE 10 Example Compound Characterization IC₅₀ (μM) 73

¹H NMR (400 MHz, DMSO-d₆) δ ppm 8.10 (d, J = 5.2 Hz, 1 H), 7.69 (s, 1H), 6.57 (d, J = 5.3 Hz, 1 H), 6.30 (s, 2 H), 4.06 (s, 2 H), 3.79-3.60(m, 4 H), 1.75-1.52 (m, 4 H). HRMS calcd for C₁₆H₁₈Cl₂N₇OS (M + H)⁺426.0665, found 426.0628. 0.020 74

¹H NMR (400 MHz, METHANOL-d₄) δ ppm 8.40 (dd, J = 4.3, 1.7 Hz, 1 H),7.64 (s, 1 H), 7.52-7.30 (m, 2 H), 4.72 (s, 2 H), 4.00 (dt, J = 14.2,5.1 Hz, 2 H), 3.54 (ddd, J = 13.5, 8.7, 3.8 Hz, 2 H), 1.97 (dtq, J =17.6, 8.6, 4.1 Hz, 4 H). HRMS calcd for C₁₇H₁₉F₃N₇OS (M + H)⁺ 426.1318,found 426.1344. 0.056

Example 75(R)-8-(6-amino-5-((3-chloro-2-(dimethylamino)pyridin-4-yl)thio)pyrazin-2-yl)-8-azaspiro[4.5]decan-1-amine

Step a: A mixture of6-bromo-3-((3-chloro-2-(dimethylamino)pyridin-4-yl)thio)pyrazin-2-amine(124 mg, 0.392 mmol) and(R)-2-methyl-N—((R)-8-azaspiro[4.5]decan-1-yl)propane-2-sulfinamide (111mg, 0.431 mmol) in DIPEA (2.6 mL) was stirred for 10 h at 90° C. Aftercooling to RT, the volatiles were removed under reduced pressure and theresulting residue was purified by silica chromatography (0 to 10%gradient of EtOAc (containing 10% of MeOH)/heptane (containing 25 ofEt₃N)) to give(R)—N—((R)-8-(6-amino-5-((3-chloro-2-(dimethylamino)pyridin-4-yl)thio)pyrazin-2-yl)-8-azaspiro[4.5]decan-1-yl)-2-methylpropane-2-sulfinamide(75 mg, 0.139 mmol). MS m/z 538.3 (M+H)⁺.

Step b: A mixture of(R)—N—((R)-8-(6-amino-5-((3-chloro-2-(dimethylamino)pyridin-4-yl)thio)pyrazin-2-yl)-8-azaspiro[4.5]decan-1-yl)-2-methylpropane-2-sulfinamide(75 mg, 0.139 mmol) and HCl (4 M in dioxane, 174 μL, 0.697 mmol) in DCM(2 mL) was stirred for 30 min at RT. The volatiles were removed underreduced pressure and the resulting residue was purified by HPLC(gradient elution 35-60% acetonitrile in water, 5 mM NH₄OH modifier) togive(R)-8-(6-amino-5-((3-chloro-2-(dimethylamino)pyridin-4-yl)thio)pyrazin-2-yl)-8-azaspiro[4.5]decan-1-amine(28 mg, 0.064 mmol) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ ppm7.85-7.92 (m, 1H), 7.63 (s, 1H), 6.16 (br. s, 2H), 6.04-6.10 (m, 1H),4.06-4.23 (i, 2H), 2.97-3.15 (i, 2H), 2.87 (s, 6H), 2.64-2.73 (i, 1H),1.11-1.97 (in, 10H). HRMS calcd for C₂₁H₂₉ClN₇S (M+H) 434.1894, found434.1883. IC₅₀ is 0.010 μM.

The following compounds of (table 11 were synthesized using the aboveprocedure or modifications to the above procedure using thecorresponding protected amine and chloro-pyrazine intermediate.

TABLE 11 Example Compound Characterization IC₅₀ (μM) 76

¹H NMR (400 MHz, DMSO-d₆) δ ppm 8.79 (d, J = 5.3 Hz, 1 H), 7.59 (s, 1H), 6.76 (d, J = 5.5 Hz, 1 H), 6.24 (s, 2 H), 4.09 (m, 2 H), 3.00 (m, 3H), 2.65 (t, J = 7.4 Hz, 1 H), 1.76 (m, 2 H), 1.50 (m, 4 H), 1.37-1.05(m, 5 H). HRMS calcd for C₁₇H₂₃ClN₇S (M + H)⁺ 392.1424, found 392.0977.0.078 77

TFA salt. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 7.94 (d, J = 5.3 Hz, 1 H),7.79 (br. s., 3 H), 7.69 (br. s., 1 H), 6.51 (d, J = 5.5 Hz, 1 H), 6.34(br. s., 2 H), 4.12-4.32 (m, 2 H), 2.99-3.24 (m, 3 H), 2.00-2.12 (m, 1H), 1.30-1.90 (m, 9 H). HRMS calcd for C₁₈H₂₃ClFN₆S (M + H)⁺ 409.1377,found 409.1385. 0.005 78

¹H NMR (400 MHz, METHANOL-d₄) δ ppm 7.70-7.87 (m, 1 H), 7.52-7.64 (m,H), 6.22 (d, J = 5.5 Hz, 1 H), 4.27 (t, J = 13.7 Hz, 2 H), 3.97 (br. s.,3 H), 3.03-3.20 (m, 2 H), 2.82 (t, J = 7.5 Hz, 1 H), 1.98-2.18 (m, 1 H),1.24-1.96 (m, 9 H). HRMS calcd for C₁₉H₂₆ClN₆OS (M + H)⁺ 421.1577, found421.1594. 0.015 79

¹H NMR (400 MHz, DMSO-d₆) δ ppm 8.49 (s, 1 H), 8.25 (d, J = 5.3 Hz, 1H), 7.66 (s, 1 H), 6.56 (d, J = 5.3 Hz, 1 H), 6.24 (s, 2 H), 4.07-4.26(m, 2 H), 2.98-3.13 (m, 2 H), 2.70 (t, J = 7.4 Hz, 1 H), 1.11-1.94 (m,10 H). HRMS calcd for C₁₈H₂₄ClN₆S (M + H)⁺ 391.1472, found 391.1480.0.023 80

¹H NMR (400 MHz, DMSO-d₆) δ ppm 8.77 (s, 1 H), 8.52 (d, J = 5.5 Hz, 1H), 7.68 (s, 1 H), 6.77 (d, J = 5.5 Hz, 1 H), 6.23 (br. s, 2 H), 4.18(t, J = 11.8 Hz, 2 H), 2.97-3.16 (m, 2 H), 2.72 (t, J = 7.5 Hz, 1 H),1.09- 1.97 (m, 10 H). HRMS calcd for C₁₉H₂₄F₃N₆S (M + H)⁺ 425.1735,found 425.1727. 0.042

Example 81(R)-4-((3-amino-5-(1-amino-8-azaspiro[4.5]decan-8-yl)pyrazin-2-yl)thio)-3-chloropyridin-2(1H)-one

A mixture of(S)—N—((R)-8-(6-amino-5-((3-chloro-2-fluoropyridin-4-yl)thio)pyrazin-2-yl)-8-azaspiro[4.5]decan-1-yl)-2-methylpropane-2-sulfinamide(17 mg, 0.033 mmol), lithium hydroxide (2 mg, 0.040 mmol), and water(0.07 mL) in DMSO (0.3 mL) was stirred in a microwave reactor for 45 minat 90° C. After cooling to RT, MeOH (0.5 mL) was added followed by HCl(4 M in dioxane, 2.0 mL, 8.0 mmol) and the resulting mixture was stirredfor 1 h at 40° C. The volatiles were removed under reduced pressure andthe resulting residue was purified by HPLC (gradient elution 15-40%acetonitrile in water, 5 mM NH₄OH modifier) to give(R)-4-((3-amino-5-(1-amino-8-azaspiro[4.5]decan-8-yl)pyrazin-2-yl)thio)-3-chloropyridin-2(1H)one(5 mg, 0.012 mmol) as a white solid. ¹H NMR (400 MHz, METHANOL-d₄) δ ppm7.53-7.61 (m, 1H), 7.19 (d, J=7.1 Hz, 1H), 5.72 (d, J=7.1 Hz, 1H), 4.26(t, J=13.1 Hz, 2H), 3.06-3.20 (m, 2H), 2.81 (t, J=7.5 Hz, 1H), 1.27-2.11(m, 10H). HRMS calcd for C₁₈H₂₄ClN₆OS (M+H)⁺407.1448, found 407.1433.IC₅₀ is 0.020 μM.

Example 82racemic-8-(6-amino-5-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrazin-2-yl)-2,2-difluoro-8-azaspiro[4.5]decan-1-amine

Step a: A solution of tert-butyl 2,2-difluoro-1-oxo-8-azaspiro[4.5]decane-8-carboxylate (220 mg, 0.76 mmol), racemic2-methylpropane-2-sulfinamide (184 mg, 1.52 mmol), and titanium(IV)ethoxide (0.640 mL, 3.0 mmol) in THF (4 mL) was stirred for 30 min at90° C. After cooling to 0° C., lithium borohydride (33 mg, 1.5 mmol) wasadded in one portion. After stirring for 30 min, the reaction mixturewas quenched by addition of MeOH. The volatiles were removed underreduce pressure. The resulting residue was diluted with brine, it wasextracted with EtOAc (4×10 mL), the combined organic phases were driedover Na₂SO₄, filtered, and concentrated under under reduced pressure.The resulting residue was purified by silica chromatography (10 to 50%gradient of EtOAc/heptane) to give tert-butyl1-(1,1-dimethylethylsulfinamido)-2,2-difluoro-8-azaspiro[4.5]decane-8-carboxylateas white powder (190 mg, 0.48 mmol). MS m/z 395.2 (M+H)⁺.

Step b: A solution of tert-butyl1-(1,1-dimethylethylsulfinamido)-2,2-difluoro-8-azaspiro[4.5]decane-8-carboxylate(190 mg, 0.48 mmol) and TFA (1 mL) in DCM (4 mL) was stirred for 20 minat 0° C. The volatiles were removed under reduced pressure to giveN-(2,2-difluoro-8-azaspiro[4.5]decan-1-yl)-2-methylpropane-2-sulfinamidewhich was used in next step without further purification.

Step c: A solution ofN-(2,2-difluoro-8-azaspiro[4.5]decan-1-yl)-2-methylpropane-2-sulfinamide(theor 0.48 mmol) and 6-chloro-3-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrazin-2-amine (148 mg, 0.480 mmol) in DIPEA(0.8 mL) was stirred for 1 h at 100° C. After cooling to RT, thevolatiles were removed under reduced pressure and the resulting residuewas purified by silica chromatography (0 to 100% gradient ofEtOAc/heptane) to giveN-(8-(6-amino-5-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrazin-2-yl)-2,2-difluoro-8-azaspiro[4.5]decan-1-yl)-2-methylpropane-2-sulfinamide (174 mg, 0.28 mmol) as anorange powder. A portion of this material was progressed to step d, theremaining material was separated by chiral chromatography (see example83).

Step d: A solution N-(8-(6-amino-5-((2-(trifluoromethyl)pyridin-3-yl)thio) pyrazin-2-yl)-2,2-difluoro-8-azaspiro[4.5]decan-1-yl)-2-methylpropane-2-sulfinamide (54 mg, 0.096 mmol) andHCl (4 M in dioxane, 0.239 mL, 0.96 mmol) in DCM (1 mL) was stirred for30 min at 40° C. After cooling to RT, the volatiles were removed underreduced pressure. This residue was triturated with MeCN to giveracemic-8-(6-amino-5-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrazin-2-yl)-2,2-difluoro-8-azaspiro[4.5]decan-1-amine(HCl salt, 38 mg, 0.075 mmol) as a pale tan powder. ¹H NMR (400 MHz,METHANOL-d₄) δ ppm 8.52-8.38 (m, 1H), 7.71 (s, 1H), 7.50-7.43 (m, 2H),4.44 (dd, J=21.0, 14.2 Hz, 2H), 3.67 (dd, J=15.1, 11.2 Hz, 1H),3.23-3.08 (m, 2H), 2.47-2.34 (m, 2H), 2.27 (dt, J=14.6, 7.4 Hz, 1H),2.01-1.88 (m, 2H), 1.75-1.54 (m, 3H). HRMS calcd for C₁₉H2₂F5N₆S(M+H)⁺461.1547, found 461.1540. IC₅₀ is 0.380 μM.

Example 83a/b (R) and(S)-8-(6-amino-5-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrazin-2-yl)-2,2-difluoro-8-azaspiro[4.5]decan-1-amine

Step a:N-(8-(6-amino-5-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrazin-2-yl)-2,2-difluoro-8-azaspiro[4.5]decan-1-yl)-2-methylpropane-2-sulfinamide(100 mg, 0.177 mmol) was further purified by chiral SFC as follows:column: WHO-1 21×250 mm, flow rate: 80 g per minute, mobile phase: 45%MeOH and 5 mM NH₄OH in CO₂, detection: mass triggered to obtain singleenantiomers R_(t) (enantiomer R): 2.6 min (44 mg, 0.078 mmol) and R_(t)(enantiomer S): 5.8 min (41 mg, 0.073 mmol).

Step b: A mixture of pure enantiomer and HCl (4 M in dioxane, 200 μL,0.8 mmol) in DCM (2 mL) was stirred for 1 h at 40° C. The volatiles wereremoved under reduced pressure and the resulting residue was trituratedwith MeCN to give the title compounds as HCl salts:

(R)-Enantiomer: ¹H NMR (400 MHz, METHANOL-d₄) δ ppm 8.46 (dd, J=3.7, 2.3Hz, 1H), 7.73 (s, 1H), 7.53-7.45 (m, 2H), 4.52-4.36 (m, 2H), 3.68 (dd,J=15.0, 11.2 Hz, 1H), 3.24-3.09 (m, 2H), 2.47-2.34 (m, 2H), 2.32-2.21(m, 1H), 2.05-1.90 (m, 2H), 1.74-1.55 (m, 3H). ¹⁹F NMR (376 MHz,METHANOL-d₄) δ−66.19, −98.51 (d, J=234.5 Hz), −101.83 (d, J=234.6 Hz).HRMS calcd for C₁₉H₂₂F₅N₆S (M+H)⁺461.1547, found 461.1540. IC₅₀ is 0.882μM.

(S)-Enantiomer: ¹H NMR (400 MHz, METHANOL-d₄) δ ppm 8.50-8.41 (m, 1H),7.70 (s, 1H), 7.47 (m, 2H), 4.52-4.35 (m, 2H), 3.67 (dd, J=15.1, 11.2Hz, 1H), 3.24-3.05 (m, 2H), 2.49-2.32 (m, 2H), 2.31-2.19 (m, 1H),2.02-1.88 (m, 2H), 1.73-1.51 (m, 3H). ¹⁹F NMR (376 MHz, METHANOL-d₄)δ−66.24, −98.47 (d, J=234.4 Hz), −101.77 (d, J=234.6 Hz). HRMS calcd forC₁₉H₂₂F₅N₆S (M+H)⁺461.1547, found 461.1541. IC₅₀ is 0.306 μM.

Example 848-(6-amino-5-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrazin-2-yl)-2-fluoro-8-azaspiro[4.5]decan-1-amine

Step a: A solution of racemic tert-butyl2-fluoro-1-oxo-8-azaspiro[4.5]decane-8-carboxylate (78 mg, 0.28 mmol),titanium(IV) ethoxide (235 μL, 1.1 mmol), and(R)-2-methylpropane-2-sulfinamide (68 mg, 0.56 mmol) in THF (1.5 mL) wasstirred for 1 h at 90° C. After cooling to 0° C., lithium borohydride(12 mg, 0.56 mmol) was added in one portion. After stirring for 30 min,the reaction mixture was quenched by addition of MeOH. The volatileswere removed under reduce pressure. The resulting residue was dilutedwith brine, it was extracted with EtOAc (4×10 mL), the combined organicphases were dried over Na₂SO₄, filtered, and concentrated under reducedpressure. The resulting residue was purified by silica chromatography (0to 50% gradient of EtOAc/heptane) to give tert-butyl1-((R)-1,1-dimethylethylsulfinamido)-2-fluoro-8-azaspiro[4.5]decane-8-carboxylate(64 mg, 0.17 mmol). MS m/z 377.3 (M+H)⁺.

Step b: A mixture of tert-butyl1-((R)-1,1-dimethylethylsulfinamido)-2-fluoro-8-azaspiro[4.5]decane-8-carboxylate(64 mg, 0.17 mmol) and TFA (1 mL) in DCM (4 mL) was stirred for 10 minat 0° C. The volatiles were removed under reduced pressure and theresulting residue was use in next step without further purification.

Step c: A mixture of the former residue and 6-chloro-3-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrazin-2-amine (51 mg, 0.17 mmol) in DIPEA(0.3 mL) was stirred for 2 h at 100° C. After cooling to RT, thevolatiles were removed under reduced pressure and the resulting residuewas purified by silica chromatography (0 to 10% gradient of MeOH/DCM(containing 0.25% Et₃N)) to giveN-(8-(6-amino-5-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrazin-2-yl)-2-fluoro-8-azaspiro[4.5]decan-1-yl)-2-methylpropane-2-sulfinamide(41 mg, 0.075 mmol). MS m/z 547.2 (M+H)⁺ as mixture of diastereomers.Further purification using chiral SFC was performed as follows: column:ID 21×250 mm, flow rate: 80 g per minute, mobile phase: 45% iPrOH and 10mM NH₄OH in CO₂, detection: mass triggered to give single enantiomersR_(t) (P1)=2.7 min (17 mg, 0.031 mmol) and R_(t) (enant-P1)=4.4 min (17mg, 0.031 mmol).

Step d: A solution of each pure isomer and HCl (4 M in dioxane, 100 μL,0.4 mmol) in DCM (0.1 mL) was stirred for 1 h at 40° C. After cooling toRT, the volatiles were removed under reduced pressure and the resultingresidue was triturated with MeCN to give the title compounds as HClsalts.

TABLE 12 Example Compound Characterization IC₅₀ (μM) 85

HCl salt. P1: ¹H NMR (400 MHz, METHANOL-d₄) δ ppm 8.45 (dd, J = 3.7, 2.3Hz, 1 H), 7.70 (s, 1 H), 7.51-7.43 (m, 2 H), 5.31-5.08 (m, 1 H), 4.46(t, J = 15.0 Hz, 2 H), 3.36 (d, J = 4.2 Hz, 1 H), 3.20- 3.06 (m, 2 H),2.28-2.06 (m, 3 H), 1.96- 1.82 (m, 2 H), 1.74-1.50 (m, 3 H). ¹⁹F NMR(376 MHz, METHANOL-d₄) δ −65.01, −66.44. HRMS calcd for C₁₉H₂₃F₄N₆S (M +H)⁺ 443.1641, found 443.1642. HCl salt. enant-P1: ¹H NMR (400 MHz,METHANOL-d₄) δ ppm 8.48-8.44 (m, 1 H), 7.71 (s, 1 H), 7.51-7.42 (m, 2H), 5.30- 5.10 (m, 1 H), 4.47 (t, J = 15.1 Hz, 2 H), 3.37-3.34 (m, 1 H),3.21-3.06 (m, 2 H), 2.28-2.08 (m, 3 H), 1.95-1.82 (m, 2 H), 1.76-1.47(m, 3 H). ¹⁹F NMR (376 MHz, METHANOL-d₄) δ −65.01, −66.42. HRMS calcdfor C₁₉H₂₃F₄N₆S (M + H)⁺ 443.1641, found 443.1633. P1 = 0.100 enant-P1 =0.113

Example 86a/b(1R)-8-(6-amino-5-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrazin-2-yl)-2-methyl-8-azaspiro[4.5]decan-1-amine

Step a: A solution of (1R)-tert-butyl1-((R)-1,1-dimethylethylsulfinamido)-2-methyl-8-azaspiro[4.5]decane-8-carboxylate(32 mg, 0.086 mmol) and TFA (0.2 mL, 2.60 mmol) in DCM (2 mL) wasstirred for 30 min at RT. The volatiles were removed under reducedpressure to give(R)-2-methyl-N-((1R)-2-methyl-8-azaspiro[4.5]decan-1-yl)propane-2-sulfinamide.MS m/z 273.0 (M+H)⁺. The crude product was used in the next step withoutfurther purification.

Step b: A mixture of(R)-2-methyl-N-((1R)-2-methyl-8-azaspiro[4.5]decan-1-yl)propane-2-sulfinamide(23 mg, 0.084 mmol),6-chloro-3-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrazin-2-amine (23mg, 0.075 mmol), and NMP (0.1 mL) in DIPEA (1 mL) was stirred for 6 h at115° C. After cooling to RT, the volatiles were removed under reducepressure to give(R)—N-((1R)-8-(6-amino-5-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrazin-2-yl)-2-methyl-8-azaspiro[4.5]decan-1-yl)-2-methylpropane-2-sulfinamideas a black oil which was used in the next step without purification.

Step c: A mixture of(R)—N-((1R)-8-(6-amino-5-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrazin-2-yl)-2-methyl-8-azaspiro[4.5]decan-1-yl)-2-methylpropane-2-sulfinamideand HCl (4 M in dioxane, 84 μL, 0.338 mmol) in DCM (2 mL) was stirredfor 1 h at 40° C. After cooling to RT, the volatiles were removed underreduced pressure and the resulting residue was purified by HPLC(gradient elution 35-60% acetonitrile in water, 0.1% TFA modifier) togive8-(6-amino-5-((2-(trifluoromethyl)pyridin-3-yl)thio)pyrazin-2-yl)-2-methyl-8-azaspiro[4.5]decan-1-amineTFA salt. HRMS calcd for C₂₀H₂₆F₃N₆S 439.1892 (M+H)⁺, found 439.1872.IC₅₀ is 0.0010 μM.

Step d: Chiral separation (see table 13 for details).

TABLE 13 IC₅₀ Example Compound Characterization (μM) 86a

Chiral SFC purification performed as follows: column: ADH 21 × 250 mm,flow rate: 80 g per minute, mobile phase: 30% MeOH and 5 mM NH₄OH inCO₂, detection: 270 nm UV to obtain a single isomer R_(t) (P1) = 3.3min. ¹H NMR (400 MHz, METHANOL-d₄) δ ppm 8.39 (dd, J = 4.4, 1.6 Hz, 1H), 7.59 (s, 1 H), 7.47- 7.35 (m, 2 H), 4.46-4.31 (m, 2 H), 3.15- 2.95(m, 2 H), 2.44 (d, J = 9.5 Hz, 1 H), 2.02-1.67 (m, 5 H), 1.61-1.36 (m, 4H), 1.12 (d, J = 6.3 Hz, 3 H). HRMS calcd for 0.010  C₂₀H₂₆F₃N₆S439.1892 (M + H)⁺, found 439.1900. 86b

Chiral SFC purification performed as follows: column: ADH 21 × 250 mm,flow rate: 80 g per minute, mobile phase: 30% MeOH and 5 mM NH₄OH inCO₂, detection: 270 nm UV to obtain a single isomer R_(t) (P2) = 4.3min. ¹H NMR (400 MHz, METHANOL-d₄) δ ppm 8.38 (dd, J = 4.4, 1.6 Hz, 1H), 7.58 (s, 1 H), 7.49- 7.31 (m, 2 H), 4.19-4.00 (m, 2 H), 3.29- 3.12(m, 2 H), 2.80 (d, J = 5.9 Hz, 1 H), 2.38-2.24 (m, 1 H), 1.91-1.47 (m, 8H), 0.010  1.01 (d, J = 7.0 Hz, 3 H). HRMS calcd for C₂₀H₂₆F₃N₆S439.1892 (M + H)⁺, found 439.1880. 87

TFA salt. ¹H NMR (400 MHz, METHANOL-d₄) δ ppm 7.59 (s, 1 H), 5.77 (s, 1H), 4.41-4.22 (m, 2 H), 3.24 (t, J = 6.8 Hz, 1 H), 3.21-3.08 (m, 2 H),2.29- 2.16 (m, 1 H), 1.97-1.79 (m, 4 H), 1.78- 1.65 (m, 2 H), 1.65-1.48(m, 3 H). HRMS calcd for C₁₈H₂₄Cl₂N₇S (M + H)⁺ 440.1191, found 440.1169.0.0093 88

TFA salt. ¹H NMR (400 MHz, METHANOL-d₄) δ ppm 7.60 (s, 1 H), 5.76 (s, 1H), 4.37-4.19 (m, 3 H), 3.98 (d, J = 9.2 Hz, 1 H), 3.88 (d, J = 9.2 Hz,1 H), 3.42 (d, J = 4.0 Hz, 1 H), 3.13 (dddd, J = 26.3, 14.0, 11.0, 3.2Hz, 2 H), 1.92-1.74 (m, 3 H), 1.73-1.63 (m, 1 H), 1.31 (d, J = 6.5 Hz, 3H). HRMS calcd for C₁₈H₂₄Cl₂N₇OS (M + H)⁺ 456.1140, found 456.1118.0.004  89

TFA salt. ¹H NMR (400 MHz, METHANOL-d₄) δ ppm 7.61 (s, 1 H), 5.77 (s, 1H) 4.35-4.14 (m, 3 H), 3.98 (d, J = 9.2 Hz, 1 H), 3.89 (d, J = 9.2 Hz, 1H), 3.82 (dd, J = 10.7, 2.6 Hz, 1 H), 3.57 (dd, J = 5.3, 2.5 Hz, 1 H),3.23 (ddd, J = 14.1, 8.8, 5.5 Hz, 1 H), 3.14 (ddd, J = 13.8, 10.7, 3.3Hz, 1 H), 1.86-1.63 (m, 4 H). HRMS calcd for C₁₇H₂₂Cl₂N₇OS (M + H)⁺442.0984, found 442.0961. 0.0118 90

TFA salt. ¹H NMR (400 MHz, METHANOL-d₄) δ ppm 7.60 (s, 1 H), 5.77 (s, 1H), 4.51-4.32 (m, 3 H), 3.47 (dd, J = 9.3, 7.2 Hz, 1 H), 3.13-2.94 (m, 2H), 2.28-2.11 (m, 2 H), 2.01 (ddd, J = 13.8, 9.5, 6.0 Hz, 1 H),1.92-1.78 (m, 2 H), 1.70 (d, J = 13.2 Hz, 1 H), 1.55-1.47 (m, 2 H). HRMScalcd for C₁₈H₂₄Cl₂N₇OS (M + H)⁺ 456.1140, found 456.1111. 0.004  91

¹H NMR (400 MHz, METHANOL-d₄) δ ppm 7.52-7.64 (m, 2 H), 5.92 (d, J =5.56 Hz, 1 H), 5.13 (td, J = 56.0, 6.69, Hz, 1 H), 4.21-4.42 (m, 2 H),3.00-3.22 (m, 2 H), 2.83 (t, J = 7.71 Hz, 1 H), 2.37-2.58 (m, 1 H),2.16-2.37 (m, 1 H), 1.62-1.92 (m, 5 H), 1.21-1.36 (m, 1 H). ¹⁹F NMR (376MHz, METHANOL-d₄) δ ppm −162.81. HRMS calcd for C₁₈H₂₄ClFN₇S (M + H)⁺424.1486, found 424.1491. 0.008  92

¹H NMR (400 MHz, METHANOL-d₄) δ ppm 7.50-7.65 (m, 2 H), 5.92 (d, J =5.56 Hz, 1 H), 5.13 (dt, J = 56 Hz, 5.94 Hz, 1 H) 4.21-4.40 (m, 2 H),2.97-3.19 (m, 3 H), 2.13-2.35 (m, 2 H), 1.70-2.00 (m, 3 H), 1.62 (td, J= 12.63, 4.29 Hz, 1 H), 1.47 (dd, J = 13.26, 2.40 Hz, 1 H), 1.33 (dd, J= 13.39, 2.27 Hz, 1 H). ¹⁹F NMR (376 MHz, METHANOL-d₄) δ ppm −166.11.HRMS calcd for C₁₈H₂₄ClFN₇S (M + H)⁺ 424.1486, found 424.1486. 0.010 

The following examples of Table 14 can be made using the above methodsand appropriate starting materials:

TABLE 14

Assays

Compounds of the invention were assessed for their ability toselectively inhibit SHP2 activity. The inhibitory properties of thecompounds of the invention described herein can be evidenced by testingin any one of the following assays.

SHP2 Allosteric Inhibition Assay

SHP2 is allosterically activated through binding ofbis-tyrosyl-phorphorylated peptides to its Src Homology 2 (SH2) domains.The latter activation step leads to the release of the auto-inhibitoryinterface of SHP2, which in turn renders the SHP2 protein tyrosinephosphatase (PTP) active and available for substrate recognition andreaction catalysis. The catalytic activity of SHP2 was monitored usingthe surrogate substrate DiFMUP in a prompt fluorescence assay format.

More specifically, the phosphatase reactions were performed at roomtemperature in 384-well black polystyrene plate, flat bottom, lowflange, non-binding surface (Corning, Cat #3575) using a final reactionvolume of 25 μL and the following assay buffer conditions: 60 mM HEPES,pH 7.2, 75 mM NaCl, 75 mM KCl, 1 mM EDTA, 0.05% P-20, 5 mM DTT.

The inhibition of SHP2 by compounds of the invention (concentrationsvarying from 0.003-100 μM) was monitored using an assay in which 0.5 nMof SHP2 was incubated with of 0.5 μM of peptide IRS1_pY1172(dPEG8)pY1222(sequence: H2N-LN(pY)IDLDLV(dPEG8)LST(pY)ASINFQK-amide) (SEQ ID NO:1).After 30-60 minutes incubation at 25° C., the surrogate substrate DiFMUP(Invitrogen, cat #D6567) was added to the reaction and incubated at 25°C. for 30 minutes. The reaction was then quenched by the addition of 5of a 160 μM solution of bpV(Phen) (Enzo Life Sciences cat #ALX-270-204).The fluorescence signal was monitored using a microplate reader(Envision, Perki-Elmer) using excitation and emission wavelengths of 340nm and 450 nm, respectively. The inhibitor dose response curves wereanalyzed using normalized IC₅₀ regression curve fitting with controlbased normalization. IC₅₀ results for compounds of the invention areshown in examples and tables 1-7, above.

p-ERK Cellular Assay

p-ERK cellular assay using the AlphaScreen® SureFire™ Phospho-ERK ½ Kit(PerkinElmer): KYSE-520 cells (30,000 cells/well) were grown in 96-wellplate culture overnight and treated with Shp2 inhibitors atconcentrations of 20, 6.6, 2.2, 0.74, 0.24, 0.08, 0.027 μM for 2 hrs at37° C. Incubations were terminated by addition of 30 μL of lysis buffer(PerkinElmer) supplied with the SureFire phospho-extracellularsignal-regulated kinase (pERK) assay kit (PerkinElmer). Samples wereprocessed according to the manufacturer's directions. The fluorescencesignal from pERK was measured in duplicate using a 2101 multilabelreader (Perkin Elmer Envision). The percentage of inhibition wasnormalized by the total ERK signal and compared with the DMSO vehiclecontrol.

Colony Formation Assay and Cell Proliferation Assay

KYSE-520 Cells (1500 cells/well) were plated onto 24-well plates in 300μL medium (RPMI-1640 containing 10% FBS, Lonza). For drug treatment,compounds of the invention at various concentrations (20, 10, 5, 2.5,1.25 μM) were added 24 hours and 5 days after cell plating. At day 11,colonies were stained with 0.2% crystal violet (MP Biomedicals) andsubsequently dissolved in 20% acetic acid for quantitation using aSpectramax reader (Thermo Scientific). In cell proliferation assay,cells (1500-cells/well) were plated onto 96-well plates in 100 μL medium(RPMI-1640 containing 10% FBS, Lonza). At day 6, 50 μL Celltiter-Gloreagent (Promega) was added, and the luminescent signal was determinedaccording to the supplier's instruction (Promega).

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims.

We claim:
 1. A compound of formula I:

in which: p is selected from 0 and 1: q is selected from 0 and 1: Y₁ isselected from CH and N; Y₂ is selected from CR₆ and N; R₁ is —XR_(1a);wherein R_(1a) is selected from C₆₋₁₀aryl, C₃₋₈cycloalkyl,C₃₋₈Cycloalkenyl and a 5-9 member heteroaryl group containing from 1 to4 heteroatoms or groups independently selected from N, C(O), 0 and S;wherein said aryl or heteroaryl of R_(1a) is substituted with 1 to 5 R₉groups independently selected from halo, amino, hydroxy, N₃, C₁₋₄alkyl,dimethyl-amino, hydroxy-substituted-C₁₋₄alkyl,halo-substituted-C₁₋₄alkyl, amino-substituted-C₁₋₄alkyl, —C(O)OR₁₀ and—NHC(O)R₁₀; and X is selected from a bond, S(O)_(m), O, C(O), COR₁₁,CR_(10a)R_(10b), NR₁₁; wherein m is selected from 0, 1 and 2; eachR_(10a) and R_(10b) is independently selected from halo and C₁₋₄alkyl;and R₁₁ is selected from hydrogen and C₁₋₄alkyl; R_(2a) and R_(2b) areindependently selected from hydrogen, C₁₋₄alkyl, C₁₋₄alkoxy, amino,hydroxy, C₃₋₈cycloalkyl and C₁₋₄alkyl-amino; R_(3a) and R_(3b) areindependently selected from halo, carbonyl, C₁₋₄alkyl, C₁₋₄alkoxy,amino, hydroxy, C₃₋₈cycloalkyl and C₁₋₄alkyl-amino; R_(4a) and R_(4b)are independently selected from hydrogen, halo, carbonyl, C₁₋₄alkyl,C₁₋₄alkoxy, amino, hydroxy, C₃₋₈Cycloalkyl and C₁₋₄alkyl-amino; R_(5a)and R_(5b) are independently selected from hydrogen, carbonyl,C₁₋₄alkyl, C₁₋₄alkoxy, amino, hydroxy, C₃₋₈cycloalkyl andC₁₋₄alkyl-amino; wherein any two groups selected from R_(2a), R_(2b),R_(3a), R_(3b), R_(4a), R_(4b), R_(5a), R_(5b) and R₇ can form a 5 to 6member unsaturated or partially saturated ring; R₆ is selected fromhydrogen, halo, cyano, C₁₋₄alkyl, C₁₋₄alkoxy, amino-carbonyl,halo-substituted C₁₋₄alkyl, halo-substituted C₁₋₄alkoxy,hydroxy-substituted C₁₋₄alkyl, amino-substituted C₁₋₄alkyl,—S(O)₁₋₂R_(6a), —C(S)R_(6a), —C(O)NR_(6a)R_(6b), —C(NH)NR_(6a)R_(6b) and—NR_(6a)C(O)R_(6b); wherein R_(6a) and R_(6b) are independently selectedfrom hydrogen and C₁₋₄alkyl; R₇ and R₈ together with the carbon atom towhich they are both attached form a 3 to 7 member saturated or partiallyunsaturated ring that can optionally contain 1 to 3 heteroatoms orgroups independently selected from N, C(O)O and S(O)m; wherein m isselected from 0, 1 and 2; wherein said saturated ring formed by R₇ andR₈ can be unsubstituted or substituted with 1 to 3 groups independentlyselected from amino, hydroxy, methoxy, halo, methyl, methyl-amino andisobutyryloxy; or a pharmaceutically acceptable salt thereof.
 2. Thecompound of claim 1 of Formula Ia:

in which: n is selected from 1, 2, 3 and 4; p is selected from 0 and 1;q is selected from 0 and 1; Y₁ is selected from CH and N; Y₂ is selectedfrom CR and N; each Y₄ is independently selected from N, C(O) and CR₉;wherein only one Y₄ is C(O); R₆ is selected from hydrogen, halo, methyland amino-carbonyl; R₇ and R₈ together with the carbon atom to whichthey are both attached form a 3 to 7 member saturated or partiallyunsaturated ring that can optionally contain a heteroatom selected fromN, O and S(O)m; wherein m is selected from 0, 1 and 2; wherein saidsaturated ring formed by R₇ and R₈ can be unsubstituted or substitutedwith 1 to 3 groups independently selected from amino, halo, hydroxy,amino-methyl and methyl-amino; R₉ is selected from halo, amino, hydroxy,N₃, dimethyl-amino, C₁₋₄alkyl, halo-substituted-C₁₋₄alkyl, C₁₋₄alkoxy,—C(O)OR₁₀ and —NHC(O)R₁₀; R₁₀ is selected from hydrogen, phenyl andnaphthyl; wherein said phenyl of R₁₀ is unsubstituted or substitutedwith methoxy; or a pharmaceutically acceptable salt thereof.
 3. Thecompound of claim 2 in which R₇ and R₈ together with the carbon atom towhich they are both attached form a 5 member saturated or partiallyunsaturated ring that can optionally contain 1 to 2 heteroatoms orgroups independently selected from N, O, C(O) and S(O)m; wherein m isselected from 0, 1 and 2; wherein said saturated ring formed by R₇ andR₈ is substituted with 1 to 3 groups independently selected from amino,hydroxy, methoxy, halo, methyl, methyl-amino and isobutyryloxy; or apharmaceutically acceptable salt thereof.
 4. The compound of claim 3, orthe pharmaceutically acceptable salt thereof, selected from:


5. The compound of claim 2 in which R₇ and R₈ together with the carbonatom to which they are both attached form a 6 member saturated orpartially unsaturated ring that can optionally contain a heteroatomselected from N, O and S(O)m; wherein n is selected from 0, 1 and 2;wherein said saturated ring formed by R₇ and R₈ is substituted withamino; or a pharmaceutically acceptable salt thereof.
 6. The compound ofclaim 5, or the pharmaceutically acceptable salt thereof selected from:


7. The compound of claim 2 in which R₇ and R₈ together with the carbonatom to which they are both attached form a 4 member saturated orpartially unsaturated ring that can optionally contain a heteroatomselected from N, O and S(O)m; wherein m is selected from 0, 1 and 2;wherein said saturated ring formed by R₇ and R₈ is substituted with agroup selected from amino, amino-methyl and methyl-amino; or apharmaceutically acceptable salt thereof.
 8. The compound of claim 7, orthe pharmaceutically acceptable salt thereof, selected from:


9. The compound of claim 2 in which p and q are both 0; or thepharmaceutically acceptable salt thereof.
 10. The compound of claim 9,or the pharmaceutically acceptable salt thereof, selected from:


11. The compound of claim 1 of formula II:

in which: p is selected from 0 and 1; q is selected from 0 and 1; Y₁ isselected from CH and N; Y₂ is selected from CR₆ and N; R₁ is selectedfrom C₆₋₁₀aryl, C₃₋₈cycloalkyl, C₃₋₈Cycloalkenyl and a 5-9 memberheteroaryl group containing from 1 to 4 heteroatoms selected from N, Oand S; wherein said aryl or heteroaryl of R_(1a) is substituted with 1to 5 R₉ groups independently selected from halo, amino, hydroxy, N₃,C₁₋₄alkyl, hydroxy-substituted-C₁₋₄alkyl, halo-substituted-C₁₋₄alkyl,amino-substituted-C₁₋₄alkyl, —C(O)OR₁₀ and —NHC(O)R₁₀; wherein m isselected from 0, 1 and 2; each R_(10a) and R_(10b) is independentlyselected from halo and C₁₋₄alkyl; and R₁₁ is selected from hydrogen andC₁₋₄alkyl; R_(2a) and R_(2b) are independently selected from hydrogen,C₁₋₄alkyl, C₁₋₄alkoxy, amino, hydroxy, C₃₋₈cycloalkyl andC₁₋₄alkyl-amino; R_(3a) and R_(3b) are independently selected from halo,carbonyl, C₁₋₄alkyl, C₁₋₄alkoxy, amino, hydroxy, C₃₋₈cycloalkyl andC₁₋₄alkyl-amino; R_(4a) and R_(4b) are independently selected fromhydrogen, halo, carbonyl, C₁₋₄alkyl, C₁₋₄alkoxy, amino, hydroxy,C₃₋₈cycloalkyl and C₁₋₄alkyl-amino; R_(5a) and R_(5b) are independentlyselected from hydrogen, carbonyl, C₁₋₄alkyl, C₁₋₄alkoxy, amino, hydroxy,C₃₋₈cycloalkyl and C₁₋₄alkyl-amino; wherein any two groups selected fromR_(2a), R_(3a), R₄, R₅, R_(6a) and R_(7a) can form a 5 to 6 memberunsaturated or partially unsaturated ring; R₆ is selected from hydrogen,halo, cyano, C₁₋₄alkyl, C₁₋₄alkoxy, amino-carbonyl, halo-substitutedC₁₋₄alkyl, halo-substituted C₁₋₄alkoxy, hydroxy-substituted C₁₋₄alkyland amino-substituted C₁₋₄alkyl; R₇ and R₈ together with the carbon atomto which they are both attached form a 3 to 7 member saturated orpartially unsaturated ring that can optionally contain a heteroatomselected from N, O and S(O)m; wherein m is selected from 0, 1 and 2;wherein said saturated ring formed by R₇ and R₈ can be unsubstituted orsubstituted with 1 to 3 groups independently selected from amino, halo,hydroxy, amino-methyl and methyl-amino; or a pharmaceutically acceptablesalt thereof.
 12. The compound of claim 11 of Formula IIa:

in which n is selected from 1, 2, 3 and 4; p is selected from 0 and 1; qis selected from 0 and 1; Y₁ is selected from CH and N; Y₂ is selectedfrom CR₆ and N; Y₄ is selected from N and CR₉; R₆ is selected fromhydrogen, halo, methyl and amino-carbonyl; R₇ and R₈ together with thecarbon atom to which they are both attached form a 3 to 7 membersaturated or partially unsaturated ring that can optionally contain aheteroatom selected from N, O and S(O)m; wherein m is selected from 0, 1and 2; wherein said saturated ring formed by R₇ and R₈ can beunsubstituted or substituted with a group selected from amino,amino-methyl and methyl-amino; R₉ is selected from halo, amino, hydroxy,N₃, C₁₋₄alkyl, halo-substituted-C₁₋₄alkyl, C₁₋₄alkoxy, —C(O)OR₁₀ and—NHC(O)R₁₀; R₁₀ is selected from hydrogen, phenyl and naphthyl; whereinsaid phenyl of R₁₀ is unsubstituted or substituted with methoxy; or apharmaceutically acceptable salt thereof.
 13. The compound of claim 12in which R₇ and R₈ together with the carbon atom to which they are bothattached form a 5 member saturated or partially unsaturated ring thatcan optionally contain a heteroatom selected from N, O and S(O)m;wherein in is selected from 0, 1 and 2; wherein said saturated ringformed by R₇ and R₈ is substituted with amino; or a pharmaceuticallyacceptable salt thereof.
 14. The compound of claim 13, or thepharmaceutically acceptable salt thereof, selected from:


15. The compound of claim 12 in which R₇ and R₈ together with the carbonatom to which they are both attached form a 6 member saturated ring thatcan optionally contain a heteroatom selected from N, O and S(O)m;wherein in is selected from 0, 1 and 2; wherein said saturated ringformed by R₇ and R₈ is substituted with amino; or a pharmaceuticallyacceptable salt thereof.
 16. The compound of claim 15, or thepharmaceutically acceptable salt thereof, selected from:


17. The compound of claim 2 in which R₇ and R₈ together with the carbonatom to which they are both attached form a 4 member saturated ring thatcan optionally contain a heteroatom selected from N, O and S(O)m;wherein in is selected from 0, 1 and 2; wherein said saturated ringformed by R₇ and R₈ is substituted with amino; or a pharmaceuticallyacceptable salt thereof.
 18. The compound of claim 17, or thepharmaceutically acceptable salt thereof, selected from:


19. The compound of claim 12 in which p and q are both 0; or thepharmaceutically acceptable salt thereof.
 20. The compound of claim 19,or the pharmaceutically acceptable salt thereof, selected from:


21. A pharmaceutical composition comprising a compound of claim 1 or apharmaceutically acceptable salt thereof, and at least onepharmaceutically acceptable carrier.
 22. A method of treatmentcomprising administering a compound of claim 1, or a pharmaceuticallyacceptable salt thereof, to a person in need of such treatment in aneffective amount for the prophylactic or therapeutic treatment of adisease or disorder which is mediated by the activity of SHP2.
 23. Themethod of claim 22, wherein the disease or disorder mediated by theactivity of SHP2 is selected from Noonan Syndrome, Leopard Syndrome,juvenile myelomonocytic leukemias, neuroblastoma, melanoma, acutemyeloid leukemia, breast cancer, esophageal cancer, lung cancer, coloncancer, head cancer, neuroblastoma, squamous-cell carcinoma of the headand neck, gastric carcinoma, anaplastic large-cell lymphoma andglioblastoma.